OsMAPK6 phosphorylation and CLG1 ubiquitylation of GW6a non-additively enhance rice grain size through stabilization of the substrate.

The chromatin modifier GRAIN WEIGHT 6a (GW6a) enhances rice grain size and yield. However, little is known about its gene network determining grain size. Here, we report that MITOGEN-ACTIVED PROTEIN KINASE 6 (OsMAPK6) and E3 ligase CHANG LI GENG 1 (CLG1) interact with and target GW6a for phosphorylation and ubiquitylation, respectively. Unexpectedly, however, in vitro and in vivo assays reveal that both of the two post-translational modifications stabilize GW6a. Furthermore, we uncover two major GW6a phosphorylation sites (serine142 and threonine186) targeted by OsMAPK6 serving an important role in modulating grain size. In addition, our genetic and molecular results suggest that the OsMAPK6-GW6a and CLG1-GW6a axes are crucial and operate in a non-additive manner to control grain size. Overall, our findings identify a previously unknown mechanism by which phosphorylation and ubiquitylation non-additively stabilize GW6a to enhance grain size, and reveal correlations and interactions of these posttranslational modifications during rice grain development.

Genome-wide association studies identified OsTMF as a gene regulating rice seed germination under salt stress.

Introduction: Salt tolerance during seed germination is an important trait for direct seeding and low-cost rice production. Nevertheless, it is still not clear how seed germination under salt stress is regulated genetically. Methods: In this study, genome-wide association studies (GWAS) were performed to decipher the genetic basis of seed germination under salt stress using 541 rice varieties collected worldwide. Results and discussion: Three quantitative trait loci (QTLs) were identified including qGRG3-1 on chromosome 3, qGRG3-2 on chromosome 5, and qGRG4 on chromosome 4. Assessment of candidate genes in these loci for their responses to salt stress identified a TATA modulatory factor (OsTMF) in qGRG3-2. The expression of OsTMF was up-regulated in both roots and shoots after exposure to salt stress, and OsTMF knockout mutants exhibited delayed seed germination under salt stress. Haplotype analysis showed that rice varieties carrying OsTMF-Hap2 displayed elevated salt tolerance during seed germination. These results provide important knowledge and resources to improve rice seed germination under salt stress in the future.

The Spartina alterniflora genome sequence provides insights into the salt-tolerance mechanisms of exo-recretohalophytes.

Spartina alterniflora is an exo-recretohalophyte Poaceae species that is able to grow well in seashore, but the genomic basis underlying its adaptation to salt tolerance remains unknown. Here, we report a high-quality, chromosome-level genome assembly of S. alterniflora constructed through PacBio HiFi sequencing, combined with high-throughput chromosome conformation capture (Hi-C) technology and Illumina-based transcriptomic analyses. The final 1.58 Gb genome assembly has a contig N50 size of 46.74 Mb. Phylogenetic analysis suggests that S. alterniflora diverged from Zoysia japonica approximately 21.72 million years ago (MYA). Moreover, whole-genome duplication (WGD) events in S. alterniflora appear to have expanded gene families and transcription factors relevant to salt tolerance and adaptation to saline environments. Comparative genomics analyses identified numerous species-specific genes, significantly expanded genes and positively selected genes that are enriched for 'ion transport' and 'response to salt stress'. RNA-seq analysis identified several ion transporter genes including the high-affinity K+ transporters (HKTs), SaHKT1;2, SaHKT1;3 and SaHKT1;8, and high copy number of Salt Overly Sensitive (SOS) up-regulated under high salt conditions, and the overexpression of SaHKT2;4 in Arabidopsis thaliana conferred salt tolerance to the plant, suggesting specialized roles for S. alterniflora to adapt to saline environments. Integrated metabolomics and transcriptomics analyses revealed that salt stress activate glutathione metabolism, with differential expressions of several genes such as γ-ECS, GSH-S, GPX, GST and PCS in the glutathione metabolism. This study suggests several adaptive mechanisms that could contribute our understanding of evolutional basis of the halophyte.

The cytokinin receptor OHK4/OsHK4 regulates inflorescence architecture in rice via an IDEAL PLANT ARCHITECTURE1/WEALTHY FARMER'S PANICLE-mediated positive feedback circuit.

Inflorescence architecture is important for rice (Oryza sativa) grain yield. The phytohormone cytokinin (CK) has been shown to regulate rice inflorescence development; however, the underlying mechanism mediated by CK perception is still unclear. Employing a forward genetic approach, we isolated an inactive variant of the CK receptor OHK4/OsHK4 gene named panicle length1, which shows decreased panicle size due to reduced inflorescence meristem (IM) activity. A 2-amino acid deletion in the long α-helix stalk of the sensory module of OHK4 impairs the homodimerization and ligand-binding capacity of the receptor, even though the residues do not touch the ligand-binding domain or the dimerization interface. This deletion impairs CK signaling that occurs through the type-B response regulator OsRR21, which acts downstream of OHK4 in controlling inflorescence size. Meanwhile, we found that IDEAL PLANT ARCHITECTURE1(IPA1)/WEALTHY FARMER'S PANICLE (WFP), encoding a positive regulator of IM development, acts downstream of CK signaling and is directly activated by OsRR21. Additionally, we revealed that IPA1/WFP directly binds to the OHK4 promoter and upregulates its expression through interactions with 2 TCP transcription factors, forming a positive feedback circuit. Altogether, we identified the OHK4-OsRR21-IPA1 regulatory module, providing important insights into the role of CK signaling in regulating rice inflorescence architecture.

Deep learning-enabled discovery and characterization of HKT genes in Spartina alterniflora.

Spartina alterniflora is a halophyte that can survive in high-salinity environments, and it is phylogenetically close to important cereal crops, such as maize and rice. It is of scientific interest to understand why S. alterniflora can live under such extremely stressful conditions. The molecular mechanism underlying its high-saline tolerance is still largely unknown. Here we investigated the possibility that high-affinity K+ transporters (HKTs), which function in salt tolerance and maintenance of ion homeostasis in plants, are responsible for salt tolerance in S. alterniflora. To overcome the imprecision and unstable of the gene screening method caused by the conventional sequence alignment, we used a deep learning method, DeepGOPlus, to automatically extract sequence and protein characteristics from our newly assemble S. alterniflora genome to identify SaHKTs. Results showed that a total of 16 HKT genes were identified. The number of S. alterniflora HKTs (SaHKTs) is larger than that in all other investigated plant species except wheat. Phylogenetically related SaHKT members had similar gene structures, conserved protein domains and cis-elements. Expression profiling showed that most SaHKT genes are expressed in specific tissues and are differentially expressed under salt stress. Yeast complementation expression analysis showed that type I members SaHKT1;2, SaHKT1;3 and SaHKT1;8 and type II members SaHKT2;1, SaHKT2;3 and SaHKT2;4 had low-affinity K+ uptake ability and that type II members showed stronger K+ affinity than rice and Arabidopsis HKTs, as well as most SaHKTs showed preference for Na+ transport. We believe the deep learning-based methods are powerful approaches to uncovering new functional genes, and the SaHKT genes identified are important resources for breeding new varieties of salt-tolerant crops.

Integrated metabolomics and lipidomics analyses suggest the temperature-dependent lipid desaturation promotes aflatoxin biosynthesis in Aspergillus flavus.

Temperature is one of the main factors affecting aflatoxin (AF) biosynthesis in Aspergillus flavus. Previous studies showed that AF biosynthesis is elevated in A. flavus at temperatures between 28°C-30°C, while it is inhibited at temperatures above 30°C. However, little is known about the metabolic mechanism underlying temperature-regulated AF biosynthesis. In this study, we integrated metabolomic and lipidomic analyses to investigate the endogenous metabolism of A. flavus across 6 days of mycelia growth at 28°C (optimal AF production) and 37°C (no AF production). Results showed that both metabolite and lipid profiles were significantly altered at different temperatures. In particular, metabolites involved in carbohydrate and amino acid metabolism were up-regulated at 37°C on the second day but down-regulated from days three to six. Moreover, lipidomics and targeted fatty acids analyses of mycelia samples revealed a distinct pattern of lipid species and free fatty acids desaturation. High degrees of polyunsaturation of most lipid species at 28°C were positively correlated with AF production. These results provide new insights into the underlying metabolic changes in A. flavus under temperature stress.

Linolenic Acid-Derived Oxylipins Inhibit Aflatoxin Biosynthesis in Aspergillus flavus through Activation of Imizoquin Biosynthesis.

Oxylipins play important signaling roles in aflatoxin (AF) biosynthesis in Aspergillus flavus. We previously showed that exogenous supply of autoxidated linolenic acid (AL) inhibited AF biosynthesis in A. flavus via oxylipins, but the molecular mechanism is still unknown. Here, we performed multiomics analyses of A. flavus grown in media with or without AL. Targeted metabolite analyses and quantitative reverse transcription (qRT)-polymerase chain reaction (PCR) showed that the imizoquin (IMQ) biosynthetic pathway was distinctly upregulated in the presence of AL. 13C-glucose labeling confirmed in parallel that the tricarboxylic acid cycle was also enhanced by AL, consistent with observed increases in mycelial growth. Moreover, we integrated thermal proteome profiling and molecular dynamics simulations to identify a potential receptor of AL; AL was found to interact with a transporter (ImqJ) located in the IMQ gene cluster, primarily through hydrophobic interactions. Further analyses of strains with an IMQ pathway transcription factor overexpressed or knocked out confirmed that this pathway was critical for AL-mediated inhibition of AF biosynthesis. Comparison of 22 assembled A. flavus and Aspergillus oryzae genomes showed that genes involved in the IMQ pathway were positively selected in A. oryzae. Taken together, the results of our study provide novel insights into oxylipin-mediated regulation of AF biosynthesis and suggest potential methods for preventing AF contamination of crops.

A group of CLE peptides regulates de novo shoot regeneration in Arabidopsis thaliana.

Known for their regulatory roles in stem cell homeostasis, CLAVATA3/ESR-RELATED (CLE) peptides also function as mediators of external stimuli such as hormones. De novo shoot regeneration, representing the remarkable plant cellular plasticity, involves reconstitution of stem cells under control of stem-cell regulators. Yet whether and how stem cell-regulating CLE peptides are implicated in plant regeneration remains unknown. By CRISPR/Cas9-induced loss-of-function studies, peptide application, precursor overexpression, and expression analyses, the role of CLE1-CLE7 peptides and their receptors in de novo shoot regeneration was studied in Arabidopsis thaliana. CLE1-CLE7 are induced by callus-induction medium and dynamically expressed in pluripotent callus. Exogenously-applied CLE1-CLE7 peptides or precursor overexpression effectively leads to shoot regeneration suppression, whereas their simultaneous mutation results in enhanced regenerative capacity, demonstrating that CLE1-CLE7 peptides redundantly function as negative regulators of de novo shoot regeneration. CLE1-CLE7-mediated shoot regeneration suppression is impaired in loss-of-function mutants of callus-expressed CLAVATA1 (CLV1) and BARELY ANY MERISTEM1 (BAM1) genes, indicating that CLV1/BAM1 are required for CLE1-CLE7-mediated shoot regeneration signaling. CLE1-CLE7 signaling resulted in transcriptional repression of WUSCHEL (WUS), a stem cell-promoting transcription factor known as a principal regulator of plant regeneration. Our results indicate that functionally-redundant CLE1-CLE7 peptides genetically act through CLV1/BAM1 receptors and repress WUS expression to modulate shoot-regeneration capacity, establishing the mechanistic basis for CLE1-CLE7-mediated shoot regeneration and a novel role for CLE peptides in hormone-dependent developmental plasticity.

Cereal Endosperms: Development and Storage Product Accumulation.

The persistent triploid endosperms of cereal crops are the most important source of human food and animal feed. The development of cereal endosperms progresses through coenocytic nuclear division, cellularization, aleurone and starchy endosperm differentiation, and storage product accumulation. In the past few decades, the cell biological processes involved in endosperm formation in most cereals have been described. Molecular genetic studies performed in recent years led to the identification of the genes underlying endosperm differentiation, regulatory network governing storage product accumulation, and epigenetic mechanism underlying imprinted gene expression. In this article, we outline recent progress in this area and propose hypothetical models to illustrate machineries that control aleurone and starchy endosperm differentiation, sugar loading, and storage product accumulations. A future challenge in this area is to decipher the molecular mechanisms underlying coenocytic nuclear division, endosperm cellularization, and programmed cell death.

Development of a method to screen and isolate xanthine oxidase inhibitors from black bean in a single step: Hyphenation of semipreparative liquid chromatography and stepwise flow rate countercurrent chromatography.

Black bean, in which isoflavones are the main active constituent, also contains saponins and monoterpenes. Soybean isoflavone is a secondary metabolite that is formed during the growth of soybean; it exhibits antioxidant and cardiovascular activities and traces estrogen-like effects. In this study, black bean isoflavones were extracted with n-butanol, and ultrafiltration-liquid chromatography-mass spectrometry was used to screen their activity. Subsequently, the inhibitors were isolated and purified using semipreparative liquid chromatography and stepwise flow rate countercurrent chromatography. Thereafter, five active compounds were identified using mass spectrometry and nuclear magnetic resonance experiments. Finally, the inhibition types of the xanthine oxidase inhibitors were determined using enzymatic kinetic studies. The IC50 values of daidzin, glycitein-7-O-glucoside, genistin, daidzein, and genistein were determined to be 35.08, 56.22, 30.76, 68.79, and 95.37 µg/mL, respectively. Daidzin, genistin, and daidzein exhibited reversible inhibition, whereas glycitein-7-O-glucoside and genistein presented irreversible inhibition. This novel approach, which was based on ultrafiltration-liquid chromatography-mass spectrometry and stepwise flow rate countercurrent chromatography, is a powerful method for screening and isolating xanthine oxidase inhibitors from complex matrices. The study of enzyme inhibition types is helpful for understanding the underlying inhibition mechanism. Therefore, a beneficial platform was developed for the large-scale production of bioactive and nutraceutical ingredients.

Rice with Multilayer Aleurone: A Larger Sink for Multiple Micronutrients.

Diet-related noncommunicable diseases impose a heavy burden on human health worldwide. Rice is a good target for diet-related disease prevention strategies because it is widely consumed. Liu et al. (Proc Natl Acad Sci USA 115(44):11327-11332, 2018. https://doi.org/10.1073/pnas.1806304115 ) demonstrated that increasing the number of cell layers and thickness of putative aleurone in ta2-1 (thick aleurone 2-1) mutant rice enhances simultaneously the content of multiple micronutrients. However, the increases of aleurone-associated nutrients were not proportional to the increases in the aleurone thickness. In this study, first, cytohistological analyses and transmission electron microscopy demonstrated that the multilayer in ta2-1 exhibited aleurone cell structural features. Second, we detected an increase in insoluble fibre and insoluble bound-phenolic compounds, a shift in aleurone-specific neutral non-starch polysaccharide profile, enhancement of phytate and minerals such as iron, zinc, potassium, magnesium, sulphur, and manganese, enrichment of triacylglycerol and phosphatidylcholine but slight reduction in free fatty acid, and an increase in oleic fatty acid composition. These findings support our hypothesis that the expanded aleurone-like layers in ta2-1 maintained some of the distinctive aleurone features and composition. We provide perspectives to achieve even greater filling of this expanded micronutrient sink to provide a means for multiple micronutrient enhancements in rice.

CLE peptides: critical regulators for stem cell maintenance in plants.

MAIN CONCLUSION: Plant CLE peptides, which regulate stem cell maintenance in shoot and root meristems and in vascular bundles through LRR family receptor kinases, are novel, complex, and to some extent conserved. Over the past two decades, peptide ligands of the CLAVATA3 (CLV3) /Embryo Surrounding Region (CLE) family have been recognized as critical short- and long-distance communication signals in plants, especially for stem cell homeostasis, cell fate determination and physiological responses. Stem cells located at the shoot apical meristem (SAM), the root apical meristem (RAM) and the procambium divide and differentiate into specialized cells that form a variety of tissues such as epidermis, ground tissues, xylem and phloem. In the SAM of Arabidopsis (Arabidopsis thaliana), the CLV3 peptide restricts the number of stem cells via leucine-rich repeat (LRR)-type receptor kinases. In the RAM, root-active CLE peptides are critical negative regulators, while ROOT GROWTH FACTOR (RGF) peptides are positive regulators in stem cell maintenance. Among those root-active CLE peptides, CLE25 promotes, while CLE45 inhibits phloem differentiation. In vascular bundles, TRACHEARY ELEMENT DIFFERENTIATION INHIBITORY FACTOR (TDIF)/CLE41/CLE44 promotes procambium cell division, and prevents xylem differentiation. Orthologs of CLV3 have been identified in liverwort (Marchantia polymorpha), tomato (Solanum lycopersicum), rice (Oryza sativa), maize (Zea mays) and lotus (Lotus japonicas), suggesting that CLV3 is an evolutionarily conserved signal in stem cell maintenance. However, functional characterization of endogenous CLE peptides and corresponding receptor kinases, and the downstream signal transduction has been challenging due to their genome-wide redundancies and rapid evolution.

The leucine-rich repeat receptor-like kinase OsERL plays a critical role in anther lobe formation in rice.

In Arabidopsis, ERECTA (ER) subfamily of leucine-rich repeat (LRR) receptor kinases (LRR-RKs) play important roles in cell division and cell elongation. However, the functions of OsER genes in rice are still very much unknown. In this study, sixty-seven TILLING and four gene-edited mutants were identified for one of the three OsERs, OsERL, and used for functional analyses. Results showed that mutations in OsERL led to striking defects in anther development. Compete male sterility and reduced numbers of anther lobes, more severe than knockout mutants, were observed in mutants with amino acid substitutions in the kinase domain. Among alleles with amino acid changes in LRRs, only one mutation in the 16th LRR showed evident phenotype, suggesting a role of the LRR in ligand sensing. OsERL is expressed in shoot apcies, internodes and anthers, and within the anther OsERL is expressed in sporophytic and tapetal cells. Cell biological analyses revealed that mutations in OsERL led to defected periclinal division in archesporial cells in anthers, suggesting a critical role of OsERL in rice anther development.

Screening anti-gout compounds from Phellinus igniarius by ultrafiltration liquid chromatography mass spectrometry based on evaluation of an in vitro method combined with enzymatic reaction.

In the present study, the anti-inflammation effect of Phellinus igniarius extract was detected on an in vitro model of RAW 264.7 cells stimulated using sodium urate. In this cell model, the content changes of inflammatory cytokines, intercellular adhesion molecule-1, and interleukin-1 beta, in cell culture supernatants were detected using an enzyme-linked immunosorbent assay. The xanthine oxidase inhibitory activity of P. igniarius extracts was determined using a microplate reader. Furthermore, in order to identify the active compounds of P. igniarius, ultrafiltration liquid chromatography mass spectrometry was utilized to screen xanthine oxidase inhibitors from the extract. Our results showed that in the presence of P. igniarius extract, the expressions of interleukin-1 beta and intercellular adhesion molecule-1 decreased (p < 0.01 and p < 0.05, respectively) compared to that in the control group. The extract effective inhibited the xanthine oxidase activity. Finally, seven compounds were screened and identified as potential xanthine oxidase inhibitors from P. igniarius. Taken together, these results demonstrate a potential anti-inflammation bioactivity of P. igniarius in vitro, providing a basis for further in vivo research for the prevention and treatment of gout.

Defective mitochondrial function by mutation in THICK ALEURONE 1 encoding a mitochondrion-targeted single-stranded DNA-binding protein leads to increased aleurone cell layers and improved nutrition in rice.

Cereal endosperm comprises an outer aleurone and an inner starchy endosperm. Although these two tissues have the same developmental origin, they differ in morphology, cell fate, and storage product accumulation, with the mechanism largely unknown. Here, we report the identification and characterization of rice thick aleurone 1 (ta1) mutant that shows an increased number of aleurone cell layers and increased contents of nutritional factors including proteins, lipids, vitamins, dietary fibers, and micronutrients. We identified that the TA1 gene, which is expressed in embryo, aleurone, and subaleurone in caryopses, encodes a mitochondrion-targeted protein with single-stranded DNA-binding activity named OsmtSSB1. Cytological analyses revealed that the increased aleurone cell layers in ta1 originate from a developmental switch of subaleurone toward aleurone instead of starchy endosperm in the wild type. We found that TA1/OsmtSSB1 interacts with mitochondrial DNA recombinase RECA3 and DNA helicase TWINKLE, and downregulation of RECA3 or TWINKLE also leads to ta1-like phenotypes. We further showed that mutation in TA1/OsmtSSB1 causes elevated illegitimate recombinations in the mitochondrial genome, altered mitochondrial morphology, and compromised energy supply, suggesting that the OsmtSSB1-mediated mitochondrial function plays a critical role in subaleurone cell-fate determination in rice.

DEAD-BOX RNA HELICASE 27 regulates microRNA biogenesis, zygote division, and stem cell homeostasis.

After double fertilization, zygotic embryogenesis initiates a new life cycle, and stem cell homeostasis in the shoot apical meristem (SAM) and root apical meristem (RAM) allows plants to produce new tissues and organs continuously. Here, we report that mutations in DEAD-BOX RNA HELICASE 27 (RH27) affect zygote division and stem cell homeostasis in Arabidopsis (Arabidopsis thaliana). The strong mutant allele rh27-1 caused a zygote-lethal phenotype, while the weak mutant allele rh27-2 led to minor defects in embryogenesis and severely compromised stem cell homeostasis in the SAM and RAM. RH27 is expressed in embryos from the zygote stage, and in both the SAM and RAM, and RH27 is a nucleus-localized protein. The expression levels of genes related to stem cell homeostasis were elevated in rh27-2 plants, alongside down-regulation of their regulatory microRNAs (miRNAs). Further analyses of rh27-2 plants revealed reduced levels of a large subset of miRNAs and their pri-miRNAs in shoot apices and root tips. In addition, biochemical studies showed that RH27 associates with pri-miRNAs and interacts with miRNA-biogenesis components, including DAWDLE, HYPONASTIC LEAVES 1, and SERRATE. Therefore, we propose that RH27 is a component of the microprocessor complex and is critical for zygote division and stem cell homeostasis.