GhHAM regulates GoPGF-dependent gland development and contributes to broad-spectrum pest resistance in cotton.

Cotton is a globally cultivated crop, producing 87% of the natural fiber used in the global textile industry. The pigment glands, unique to cotton and its relatives, serve as a defense structure against pests and pathogens. However, the molecular mechanism underlying gland formation and the specific role of pigment glands in cotton's pest defense are still not well understood. In this study, we cloned a gland-related transcription factor GhHAM and generated the GhHAM knockout mutant using CRISPR/Cas9. Phenotypic observations, transcriptome analysis, and promoter-binding experiments revealed that GhHAM binds to the promoter of GoPGF, regulating pigment gland formation in cotton's multiple organs via the GoPGF-GhJUB1 module. The knockout of GhHAM significantly reduced gossypol production and increased cotton's susceptibility to pests in the field. Feeding assays demonstrated that more than 80% of the cotton bollworm larvae preferred ghham over the wild type. Furthermore, the ghham mutants displayed shorter cell length and decreased gibberellins (GA) production in the stem. Exogenous application of GA3 restored stem cell elongation but not gland formation, thereby indicating that GhHAM controls gland morphogenesis independently of GA. Our study sheds light on the functional differentiation of HAM proteins among plant species, highlights the significant role of pigment glands in influencing pest feeding preference, and provides a theoretical basis for breeding pest-resistant cotton varieties to address the challenges posed by frequent outbreaks of pests.

The dual role of GoPGF reveals that the pigment glands are synthetic sites of gossypol in aerial parts of cotton.

Gossypol and the related terpenoids are stored in the pigment gland to protect cotton plants from biotic stresses, but little is known about the synthetic sites of these metabolites. Here, we showed that GoPGF, a key gene regulating gland formation, was expressed in gland cells and roots. The chromatin immunoprecipitation sequencing (ChIP-seq) analysis demonstrated that GoPGF targets GhJUB1 to regulate gland morphogenesis. RNA-sequencing (RNA-seq) showed high accumulation of gossypol biosynthetic genes in gland cells. Moreover, integrated analysis of the ChIP-seq and RNA-seq data revealed that GoPGF binds to the promoter of several gossypol biosynthetic genes. The cotton callus overexpressing GoPGF had dramatically increased the gossypol levels, indicating that GoPGF can directly activate the biosynthesis of gossypol. In addition, the gopgf mutant analysis revealed the existence of both GoPGF-dependent and -independent regulation of gossypol production in cotton roots. Our study revealed that the pigment glands are synthetic sites of gossypol in aerial parts of cotton and that GoPGF plays a dual role in regulating gland morphogenesis and gossypol biosynthesis. The study provides new insights for exploring the complex relationship between glands and the metabolites they store in cotton and other plant species.

Efficient genome editing in cotton using the virus-mediated CRISPR/Cas9 and grafting system.

KEY MESSAGE: The extensive application of CRISPR in cotton was limited due to the labor-intensive transformation process. Thus, we here established a convenient method of CRISPR in cotton by CLCrV-mediated sgRNA delivery.

Identification of NHXs in Gossypium species and the positive role of GhNHX1 in salt tolerance.

BACKGROUND: Plant Na+/H+ antiporters (NHXs) are membrane-localized proteins that maintain cellular Na+/K+ and pH homeostasis. Considerable evidence highlighted the critical roles of NHX family in plant development and salt response; however, NHXs in cotton are rarely studied. RESULTS: The comprehensive and systematic comparative study of NHXs in three Gossypium species was performed. We identified 12, 12, and 23 putative NHX proteins from G. arboreum, G. raimondii, and G. hirsutum, respectively. Phylogenetic study revealed that repeated polyploidization of Gossypium spp. contributed to the expansion of NHX family. Gene structure analysis showed that cotton NHXs contain many introns, which will lead to alternative splicing and help plants to adapt to high salt concentrations in soil. The expression changes of NHXs indicate the possible differences in the roles of distinct NHXs in salt response. GhNHX1 was proved to be located in the vacuolar system and intensively induced by salt stress in cotton. Silencing of GhNHX1 resulted in enhanced sensitivity of cotton seedlings to high salt concentrations, which suggests that GhNHX1 positively regulates cotton tolerance to salt stress. CONCLUSION: We characterized the gene structure, phylogenetic relationship, chromosomal location, and expression pattern of NHX genes from G. arboreum, G. raimondii, and G. hirsutum. Our findings indicated that the cotton NHX genes are regulated meticulously and differently at the transcription level with possible alternative splicing. The tolerance of plants to salt stress may rely on the expression level of a particular NHX, rather than the number of NHXs in the genome. This study could provide significant insights into the function of plant NHXs, as well as propose promising candidate genes for breeding salt-resistant cotton cultivars.

The gland localized CGP1 controls gland pigmentation and gossypol accumulation in cotton.

Pigment glands, also known as black glands or gossypol glands, are specific for Gossypium spp. These glands strictly confine large amounts of secondary metabolites to the lysigenous cavity, leading to the glands' intense colour and providing defence against pests and pathogens. This study performed a comparative transcriptome analysis of glanded versus glandless cotton cultivars. Twenty-two transcription factors showed expression patterns associated with pigment glands and were characterized. Phenotypic screening of the genes, via virus-induced gene silencing, showed an apparent disappearance of pigmented glands after the silencing of a pair of homologous MYB-encoding genes in the A and D genomes (designated as CGP1). Further study showed that CGP1a encodes an active transcription factor, which is specifically expressed in the gland structure, while CGP1d encodes a non-functional protein due to a fragment deletion, which causes premature termination. RNAi-mediated silencing and CRISPR knockout of CGP1 in glanded cotton cultivars generated a glandless-like phenotype, similar to the dominant glandless mutant Gl2e . Microscopic analysis showed that CGP1 knockout did not affect gland structure or density, but affected gland pigmentation. The levels of gossypol and related terpenoids were significantly decreased in cgp1 mutants, and a number of gossypol biosynthetic genes were strongly down-regulated. CGP1 is located in the nucleus where it interacts with GoPGF, a critical transcription factor for gland development and gossypol synthesis. Our data suggest that CGP1 and GoPGF form heterodimers to control the synthesis of gossypol and other secondary metabolites in cotton.

Calcium-dependent protein kinases in cotton: insights into early plant responses to salt stress.

BACKGROUND: Soil salinization is one of the major environmental constraints to plant growth and agricultural production worldwide. Signaling components involving calcium (Ca2+) and the downstream calcium-dependent protein kinases (CPKs) play key roles in the perception and transduction of stress signals. However, the study of CPKs in cotton and their functions in response to salt stress remain unexplored. RESULTS: A total of 98 predicted CPKs were identified from upland cotton (Gossypium hirsutum L. 'TM-1'), and phylogenetic analyses classified them into four groups. Gene family distribution studies have revealed the substantial impacts of the genome duplication events to the total number of GhCPKs. Transcriptome analyses showed a wide distribution of CPKs' expression among different organs. A total of 19 CPKs were selected for their rapid responses to salt stress at the transcriptional level, most of which were also incduced by the thylene-releasing chemical ethephon, suggesting a partal overlap of the salinity and ethylene responses. Silencing of 4 of the 19 CPKs (GhCPK8, GhCPK38, GhCPK54, and GhCPK55) severely compromised the basal cotton resistance to salt stress. CONCLUSIONS: Our genome-wide expression analysis of CPK genes from up-land cotton suggests that CPKs are involved in multiple developmental responses as well as the response to different abiotic stresses. A cluster of the cotton CPKs was shown to participate in the early signaling events in cotton responses to salt stress. Our results provide significant insights on functional analysis of CPKs in cotton, especially in the context of cotton adaptions to salt stress.

Facile Cu(ii)-mediated conjugation of thioesters and thioacids to peptides and proteins under mild conditions.

The bioconjugation of peptide derivatives such as polypeptides, peptide-based probes and proteins is a vibrant area in many scientific fields. However, reports on metal-mediated chemical methods towards native peptides especially non-engineering protein modification under mild conditions are still limited. Herein, we describe a novel Cu(ii)-mediated strategy for the conjugation of thioesters/thioacids to peptides under mild conditions with high functional group tolerance. Based on this strategy, polypeptides, even peptide-based fluorescent probes, can be efficiently constructed. Finally, the selective modification of lysine residues of native Ub with thioesters could be realized and complete conjugation of Ub could be achieved even under equivalent Cu(ii). These promising results could greatly expand Cu(ii)-mediated reaction strategies on chemical biology and molecular imaging.

Mucroniferanines A-G, Isoquinoline Alkaloids from Corydalis mucronifera.

Five pairs of isoquinoline alkaloid enantiomers, mucroniferanines A-E (1-5), two inseparable epimeric pairs, mucroniferanines F and G (6, 7), and 10 known isoquinoline alkaloids (8-17) were obtained from Corydalis mucronifera. The structures were characterized using spectroscopic data analysis, and the absolute configurations were established by ECD and X-ray data analysis. The new compounds except for 3 possess a rare 9-methyl group in the isoquinoline alkaloids, and compounds 2 and 3 possess rare benzo[1,2-d:3,4-d]bis[1,3]dioxole moieties. It is the first report of stereoisomerism involving the 9-methyl phthalideisoquinoline alkaloids. Compounds (-)-4, 6, and 7 exhibited acetylcholinesterase inhibitory activities with IC50 values of 28.3, 12.2, and 11.3 µM, respectively.

Genome-wide identification and expression analysis of stress-associated proteins (SAPs) containing A20/AN1 zinc finger in cotton.

Stress-associated proteins (SAPs) containing the A20/AN1 zinc-finger domain play important roles in response to both biotic and abiotic stresses in plants. Nevertheless, few studies have focused on the SAP gene family in cotton. To explore the distributions and expression patterns of these genes, we performed genome-wide identification and characterization of SAPs in tetraploid Gossypium hirsutum L. TM-1 (AD1). A total of 37 genes encoding SAPs were identified, 36 of which were duplicated in the A and D sub-genomes. The analysis of gene architectures and conserved protein motifs revealed that nearly all A20-AN1-type SAPs were intron-free, whereas AN1-AN1-type SAPs contained one intron. The cis-elements of the SAP promoters were studied, as were the expression levels of cotton SAP genes under different stresses based on RNA-seq data and validated by qRT-PCR. Most cotton SAP genes were induced by multiple stresses and phytohormones, particularly salt stress, indicating that SAP genes may play important roles in cotton's response to unfavorable environmental changes. Among these identified SAPs, the expression of GhSAP17A/D is suppressed in cotton response to Vertillium dahliae, and the GhSAP17A/D-silenced cotton exhibits more resistance to V. dahliae. This study provides insight into the evolution of SAP genes in upland cotton and may aid in efforts at further functional identification of A20/AN1-type proteins and cotton's response to different stresses.

Direct Enol Ether Metalation-Negishi Coupling Strategy To Prepare α-Heteroaryl Enol Ethers.

A robust direct enol ether metalation-Negishi coupling using heteroaryl halides catalyzed by the palladium-Cy-DPEPhos system is reported. This method, which was demonstrated with a broad substrate scope, is a highly complementary method to the existing Heck coupling of synthesizing challenging α-heteroaryl-α-alkoxy alkenes.

The Na+/H+ antiporter GbSOS1 interacts with SIP5 and regulates salt tolerance in Gossypium barbadense.

Cotton is a globally cultivated economic crop and is a major source of natural fiber and edible oil. However, cotton production is severely affected by salt stress. Although Salt Overly Sensitive 1 (SOS1) is a well-studied Na+/H+ antiporter in multiple plant species, little is known about its function and regulatory mechanism in cotton. Here, we cloned a salt-induced SOS1 from sea-island cotton. Real-time quantitative PCR analysis revealed that GbSOS1 was induced by multiple stresses and phytohormones. Silencing GbSOS1 through virus-induced gene silencing significantly reduced cotton resistance to high Na+ but mildly affected Li+ tolerance. On the other hand, overexpression of GbSOS1 enhanced salt tolerance in yeast, Arabidopsis, and cotton largely due to the ability to maintain Na+ homeostasis in protoplasts. Yeast-two-hybrid assays and bimolecular fluorescence complementation identified a novel protein interacting with GbSOS1 on the plasma membrane, which we named SOS Interaction Protein 5 (SIP5). We found that the SIP5 gene encoded an unknown protein localized on the cell membrane. Silencing SIP5 significantly increased cotton tolerance to salt, exhibited by less wilting and plant death under salt stress. Our results revealed that GbSOS1 is crucial for cotton survival in saline soil, and SIP5 is a potentially negative regulator of SOS1-mediated salt tolerance in cotton. Overall, this study provides a theoretical basis for elucidating the molecular mechanism of SOS1, and a candidate gene for breeding salt-tolerant crops.

Comparative proteomic analysis identified proteins and the phenylpropanoid biosynthesis pathway involved in the response to ABA treatment in cotton fiber development.

Abscisic acid (ABA) is a plant hormone that plays an important role in cotton fiber development. In this study, the physiological changes and proteomic profiles of cotton (Gossypium hirsutum) ovules were analyzed after 20 days of ABA or ABA inhibitor (ABAI) treatment. The results showed that compared to the control (CK), the fiber length was significantly decreased under ABA treatment and increased under ABAI treatment. Using a tandem mass tags-based quantitative technique, the proteomes of cotton ovules were comprehensively analyzed. A total of 7321 proteins were identified, of which 365 and 69 differentially accumulated proteins (DAPs) were identified in ABA versus CK and ABAI versus CK, respectively. Specifically, 345 and 20 DAPs were up- and down-regulated in the ABA group, and 65 and 4 DAPs were up- and down-regulated in the ABAI group, respectively. The DAPs in the ABA group were mainly enriched in the biosynthesis of secondary metabolites, phenylpropanoid biosynthesis and flavonoid secondary metabolism, whereas the DAPs in the ABAI group were mainly enriched in the indole alkaloid biosynthesis and phenylpropanoid biosynthesis pathways. Moreover, 9 proteins involved in phenylpropanoid biosynthesis were upregulated after ABA treatment, suggesting that this pathway might play important roles in the response to ABA, and 3 auxin-related proteins were upregulated, indicating that auxin might participate in the regulation of fiber development under ABAI treatment.

Profile of cotton flavonoids: Their composition and important roles in development and adaptation to adverse environments.

Cotton is a commercial crop that is cultivated in more than 50 countries. The production of cotton has severely diminished in recent years owing to adverse environments. Thus, it is a high priority of the cotton industry to produce resistant cultivars to prevent diminished cotton yields and quality. Flavonoids comprise one of the most important groups of phenolic metabolites in plants. However, the advantage and biological roles of flavonoids in cotton have yet not been studied in depth. In this study, we performed a widely targeted metabolic study and identified 190 flavonoids in cotton leaves that span seven different classes with flavones and flavonols as the dominant groups. Furthermore, flavanone-3-hydroxylase was cloned and silenced to knock down flavonoid production. The results show that the inhibition of flavonoid biosynthesis affects the growth and development of cotton and causes semi-dwarfing in cotton seedlings. We also revealed that the flavonoids contribute to cotton defense against ultraviolet radiation and Verticillium dahliae. Moreover, we discuss the promising role of flavonoids in cotton development and defense against biotic and abiotic stresses. This study provides valuable information to study the variety and biological functions of flavonoids in cotton and will help to profile the advantages of flavonoids in cotton breeding.

[Normal light and fluorescence microscopy for authentication of Delphinii Brunoniani Herba of Tibet].

Dried herb of Delphinium brunonianum Royle (Ranunculaceae) has long been used under the herbal name "Xiaguobei" (Delphinii Brunoniani Herba) in traditional Tibetan medicine and prescribed for the treatment of influenza, itchy skin rash and snake bites. In order to find a useful and convenient method for the identification of microscopic features, the technique of fluorescence microscopy was applied to authenticate "Xiaguobei" of Tibet. The transverse sections of stem and leaf, as well as the powder of "Xiaguobei" were observed to seek for typical microscopic features by normal light and fluorescence microscopy. A style-like, single-cell glandular hair containing yellow secretions on the leaf, young stem and sepal of "Xiaguobei" was found. Under the fluorescence microscope, the xylem and pericycle fiber group emitted significant fluorescence. This work indicated that fluorescence microscopy could be an useful additional method for the authentication work. Without the traditional dyeing methods, the main microscopic features could be easily found by fluorescence microscopy. The results provided reliable references for the authentication of "Xiaguobei".

Screening and identification of α-glucosidase inhibitors from Cyclocarya paliurus leaves by ultrafiltration coupled with liquid chromatography-mass spectrometry and molecular docking.

Cyclocarya paliurus, as an important edible and medicinal product, has shown a good prospect in the prevention of diabetes mellitus (DM). However, it is unclear which active compounds derived from C. paliurus play a significant role in inhibiting α-glucosidase activity. In present study, affinity-based screening assay was developed to screen and identify potential α-glucosidase inhibitors from C. paliurus leaves based on affinity ultrafiltration coupled with ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS/MS) and molecular docking. After being enriched by D-101 macroporous resin, five eluent fractions with different polarity were obtained and their inhibitory activities on α-glucosidase were evaluated by an enzyme inhibition assay in vitro. The result showed that 70% ethanol fraction of C. paliurus leaves exhibited remarkable α-glucosidase inhibitory activity with the IC50 value of 17.81 µg/mL. The 70% ethanol fraction was incubated with α-glucosidase and then active compounds would form enzyme-inhibitor complexes. The complexes could be separated from inactive components by the interception ability of ultrafiltration membrane under centrifugation. A total of 36 active compounds were screened from C. paliurus leaves and the chemical structures were further characterized by UPLC-QTOF-MS/MS. Furthermore, molecular docking was performed to investigate possible inhibitory mechanisms between active compounds and α-glucosidase. The docking result showed that cyclocarioside I, pterocaryoside B, arjunolic acid, cyclocarioside Z5, cypaliuruside D and cyclocarioside N could be embedded well into the active pocket of α-glucosidase, and had significant affinity interactions with critical amino acid residues by forming hydrogen bonds, hydrophobic interactions and van der Waals, and affinity energies ranged from -9.3 to -6.7 kJ/mol. The results indicated that the developed method is rapid and effective for high throughput screening of potential α-glucosidase inhibitors from complex mixtures. Moreover, C. paliurus exhibited a remarkable inhibitory activity on α-glucosidase, making it a promising candidate for the prevention of DM.

Towards n-type conductivity in hexagonal boron nitride.

Asymmetric transport characteristic in n- and p-type conductivity has long been a fundamental difficulty in wide bandgap semiconductors. Hexagonal boron nitride (h-BN) can achieve p-type conduction, however, the n-type conductivity still remains unavailable. Here, we demonstrate a concept of orbital split induced level engineering through sacrificial impurity coupling and the realization of efficient n-type transport in 2D h-BN monolayer. We find that the O 2pz orbital has both symmetry and energy matching to the Ge 4pz orbital, which promises a strong coupling. The introduction of side-by-side O to Ge donor can effectively push up the donor level by the formation of another sacrificial deep level. We discover that a Ge-O2 trimer brings the extremely shallow donor level and very low ionization energy. By low-pressure chemical vapor deposition method, we obtain the in-situ Ge-O doping in h-BN monolayer and successfully achieve both through-plane (~100 nA) and in-plane (~20 nA) n-type conduction. We fabricate a vertically-stacked n-hBN/p-GaN heterojunction and show distinct rectification characteristics. The sacrificial impurity coupling method provides a highly viable route to overcome the n-type limitation of h-BN and paves the way for the future 2D optoelectronic devices.

Colorful Conductive Threads for Wearable Electronics: Transparent Cu-Ag Nanonets.

Electronic textiles have been regarded as the basic building blocks for constructing a new generation of wearable electronics. However, the electronization of textiles often changes their original properties such as color, softness, glossiness, or flexibility. Here a rapid room-temperature fabrication method toward conductive colorful threads and fabrics with Ag-coated Cu (Cu-Ag) nanonets is demonstrated. Cu-Ag core-shell nanowires are produced through a one-pot synthesis followed by electroless deposition. According to the balance of draining and entraining forces, a fast dip-withdraw process in a volatile solution is developed to tightly wrap Cu-Ag nanonets onto the fibers of thread. The modified threads are not only conductive, but they also retain their original features with enhanced mechanical stability and dry-wash durability. Furthermore, various e-textile devices are fabricated such as a fabric heater, touch screen gloves, a wearable real-time temperature sensor, and warm fabrics against infrared thermal dissipation. These high quality and colorful conductive textiles will provide powerful materials for promoting next-generation applications in wearable electronics.

Built-in electric field for photocatalytic overall water splitting through a TiO2/BiOBr P-N heterojunction.

Photocatalytic overall water splitting to simultaneously obtain abundant hydrogen and oxygen is still the mountain that stands in the way for the practical applications of hydrogen energy, in which composite semiconductor photocatalysts are critical for providing both electrons and holes to promote the following redox reaction. However, the interface between different components forms a deplete layer to hinder the charge transfer to a large extent. In order to enhance the charger transfer from an interface to the surface and promote the spatial separation of electron-hole pairs, a built-in electric field induced by a p-n heterojunction emerges as the best choice. As a touchstone, a p-n heterojunction of TiO2/BiOBr with a strong built-in electric field has been constructed, which presents a wide spectrum response owing to its interleaved band gaps after composition. The built-in electric field greatly enhances the separation and transportation of photogenerated carriers, resulting in fluorescence quenching due to the carrier recombination. The sample also displayed exceptional photoelectron responses: its photocurrent density (43.3 µA cm-2) was over 10 times that of TiO2 (3.5 µA cm-2) or BiOBr (4.2 µA cm-2). In addition, the sample with a molar ratio of 3 : 1 between TiO2 and BiOBr showed the best photocatalytic overall water splitting performance under visible light (λ > 420 nm): the hydrogen and oxygen production rate were 472.7 µmol gcat.-1 h-1 and 95.7 µmol gcat.-1 h-1, respectively, which are the highest values under visible light without other cocatalysts to have been reported in literature for the photocatalyst.