Regulation of Glandular Size and Phytoalexin Biosynthesis by a Negative Feedback Loop in Cotton.

Plants have evolved diverse defense mechanisms encompassing physical and chemical barriers. Cotton pigment glands are known for containing various defense metabolites, but the precise regulation of gland size to modulate defense compound levels remains enigmatic. Here, it is discovered that the VQ domain-containing protein JAVL negatively regulates pigment gland size and the biosynthesis of defense compounds, while the MYC2-like transcription factor GoPGF has the opposite effect. Notably, GoPGF directly activates the expression of JAVL, whereas JAVL suppresses GoPGF transcription, establishing a negative feedback loop that maintains the expression homeostasis between GoPGF and JAVL. Furthermore, it is observed that JAVL negatively regulates jasmonate levels by inhibiting the expression of jasmonate biosynthetic genes and interacting with GoPGF to attenuate its activation effects, thereby maintaining homeostatic regulation of jasmonate levels. The increased expression ratio of GoPGF to JAVL leads to enlarged pigment glands and elevated jasmonates and defense compounds, enhancing insect and pathogen resistance in cotton. These findings unveil a new mechanism for regulating gland size and secondary metabolites biosynthesis, providing innovative strategies for strengthening plant defense.

Single-cell RNA sequencing reveals a hierarchical transcriptional regulatory network of terpenoid biosynthesis in cotton secretory glandular cells.

Plants can synthesize a wide range of terpenoids in response to various environmental cues. However, the specific regulatory mechanisms governing terpenoid biosynthesis at the cellular level remain largely elusive. In this study, we employed single-cell RNA sequencing to comprehensively characterize the transcriptome profile of cotton leaves and established a hierarchical transcriptional network regulating cell-specific terpenoid production. We observed substantial expression levels of genes associated with the biosynthesis of both volatile terpenes (such as ß-caryophyllene and ß-myrcene) and non-volatile gossypol-type terpenoids in secretory glandular cells. Moreover, two novel transcription factors, namely GoHSFA4a and GoNAC42, are identified to function downstream of the Gossypium PIGMENT GLAND FORMATION genes. Both transcription factors could directly regulate the expression of terpenoid biosynthetic genes in secretory glandular cells in response to developmental and environmental stimuli. For convenient retrieval of the single-cell RNA sequencing data generated in this study, we developed a user-friendly web server . Our findings not only offer valuable insights into the precise regulation of terpenoid biosynthesis genes in cotton leaves but also provide potential targets for cotton breeding endeavors.

A dirigent protein complex directs lignin polymerization and assembly of the root diffusion barrier.

Functionally similar to the tight junctions present in animal guts, plant roots have evolved a lignified Casparian strip as an extracellular diffusion barrier in the endodermis to seal the root apoplast and maintain nutrient homeostasis. How this diffusion barrier is structured has been partially defined, but its lignin polymerization and assembly steps remain elusive. Here, we characterize a family of dirigent proteins (DPs) essential for both the localized polymerization of lignin required for Casparian strip biogenesis in the cell wall and for attachment of the strip to the plasma membrane to seal the apoplast. We reveal a Casparian strip lignification mechanism that requires cooperation between DPs and the Schengen pathway. Furthermore, we demonstrate that DPs directly mediate lignin polymerization as part of this mechanism.

[Response of Organic Carbon Loss to Soil Erosion and Its Drivers: A Meta-analysis].

Soil erosion is the main driving force of soil organic carbon (SOC) loss and plays an important role in the global carbon cycle. It is helpful to understand the mechanism of SOC loss under soil erosion by evaluating the main driving factors of SOC loss under soil erosion and their influence degree. Therefore, based on 24 cases published in domestic and foreign journals from 2007 to 2021, this study investigated the effects of soil erosion on SOC loss in China under different climatic factors (climate types, rainfall, and rainfall intensity) and soil factors (soil types, bulk density, and aggregate size) by using Meta-analysis. The results showed that:① compared with that under no erosion disturbance, the SOC content under erosion decreased significantly (overall decreased 16.0%), showing obvious negative response characteristics. ② Under the erosion background, the negative response degree of SOC to different factors was as follows:rainfall intensity (65.0%)>mean annual rainfall (24.3%)>soil types (21.4%)>bulk density (20.2%)>aggregate size (16.5%)>climate types (9.1%). ③ Principal component analysis showed that climate was the dominant factor affecting SOC loss, and rainfall intensity was again shown to be the key factor. In this study, the characteristics and influencing factors of SOC loss under soil erosion in China were analyzed, which provided theoretical reference for the systematic understanding of the role of soil erosion in the carbon cycle.

Dirigent gene editing of gossypol enantiomers for toxicity-depleted cotton seeds.

Axial chirality of biaryls can generate varied bioactivities. Gossypol is a binaphthyl compound made by cotton plants. Of its two axially chiral isomers, (-)-gossypol is the bioactive form in mammals and has antispermatogenic activity, and its accumulation in cotton seeds poses health concerns. Here we identified two extracellular dirigent proteins (DIRs) from Gossypium hirsutum, GhDIR5 and GhDIR6, which impart the hemigossypol oxidative coupling into (-)- and (+)-gossypol, respectively. To reduce cotton seed toxicity, we disrupted GhDIR5 by genome editing, which eliminated (-)-gossypol but had no effects on other phytoalexins, including (+)-gossypol, that provide pest resistance. Reciprocal mutagenesis identified three residues responsible for enantioselectivity. The (-)-gossypol-forming DIRs emerged later than their enantiocomplementary counterparts, from tandem gene duplications that occurred shortly after the cotton genus diverged. Our study offers insight into how plants control enantiomeric ratios and how to selectively modify the chemical spectra of cotton plants and thereby improve crop quality.

Acupoint Autohemotherapy Attenuates Atopic Dermatitis Lesions by Regulating Th1/Th2 Balance in DNCB-Induced BALB/c Mice.

OBJECTIVE: To evaluate the therapeutic effects of acupoint autohemotherapy (A-AHT) on 1-chloro-2,4-dinitrobenzene (DNCB)-induced atopic dermatitis (AD) in mice focusing on regulating T helper 1/T helper 2 (Th1/Th2) immune responses. METHODS: Thirty BALB/c mice were divided into 5 groups by a random number table, including normal control (NC), AD model (AD), A-AHT, sham A-AHT (sA-AHT), and acupoint injection of normal saline (A-NS) groups, 6 mice per group. Mice were challenged by DNCB for the establishment of experimental AD model. On the 8th day, except for the NC and AD groups, the mice in the other groups received management once every other day for a total of 28 days. For the A-AHT and sA-AHT groups, 0.05 mL of autologous whole blood (AWB) was injected into bilateral Zusanli (ST 36) and Quchi (LI 11) and sham-acupoints (5 mm lateral to ST 36 and LI 11), respectively. The A-NS group was administrated with 0.05 mL of normal saline by acupoint injection into ST 36 and LI 11. Dermatitis severity for dorsal skin of mice was determined using the Severity Scoring of Atopic Dermatitis (SCORAD) every week. The total immunoglobulin E (IgE), interleukin-4 (IL-4), and interferon-gamma (IFN-γ) cytokine levels in serum were examined by enzyme-linked immunosorbent assay (ELISA). Spleen Th1/Th2 expression were analyzed via flow cytometry and immunohistochemical assay was used to detect T-box expressed in T cell (T-bet) and GATA-binding protein 3 (GATA3) expressions in skin lesions of mice. RESULTS: Compared with the AD group, both A-AHT and sA-AHT reduced the SCORAD index and serum IgE level (P<0.05 or P<0.01); A-AHT, sA-AHT and A-NS down-regulated serum IL-4 level and upregulated IFN-γ/IL-4 ratio (P<0.05 or P<0.01); A-AHT regulated the Th1/Th2 shift specifically and increased the related transcription factors such as T-bet expression and T-bet/GATA3 ratio (P<0.05). CONCLUSION: A-AHT showed significant effectiveness on the AD model mice, through regulating Th1/Th2 immune responses.

1,10/1,11-Cyclization catalyzed by diverged plant sesquiterpene synthases is dependent on a single residue.

The exquisite chemodiversity of terpenoids is the product of the large diverse terpene synthase (TPS) superfamily. Here, by using structural and phylogenetic analyses and site-directed mutagenesis, we identified a residue (Cys440 in Nicotiana tabacum 5-epi-aristolochene synthase) proximal to an ion-binding motif common to all TPSs and named the preNSE/DTE residue, which determines the product specificity of sesquiterpene synthases from different plant species. In sesquiterpene synthases catalyzing 1,10-cyclization (1,10-cyclases) of farnesyl diphosphate, mutation of the residue in both specific and promiscuous 1,10-cyclases from different lineages leads to the accumulation of monocyclic germacrene A-11-ol, which is "short-circuited" from complex cyclization cascades, suggesting a key role of this residue in generating the first common intermediate of 1,10-cyclization. Altering this residue in a specific 1,11-cyclase results in alternative 1,10-cyclization products. Moreover, the preNSE/DTE residue can be harnessed to engineer highly specific sesquiterpene synthases for an improved proportion of high-value terpenoids, such as patchoulol, a main constituent of several traditional Chinese medicines that could treat SARS-CoV-2.

Side Products of Recombinant Amorpha-4,11-diene Synthase and Their Effect on Microbial Artemisinin Production.

Amorpha-4,11-diene synthase (ADS) is the first committed enzyme in the biosynthesis of artemisinin. Artemisinin production by biobased fermentation is considered a reliable alternative pathway. Heterologously expressed ADS has been established to generate several minor products, including structural analogues of amorpha-4,11-diene, but their fate in fermentation is still unknown. Here, using chiral analysis, we found that ADS produces one of the analogues, amorpha-4-en-11-ol, as a pair of epimers. Labeling experiments revealed that ADS mutants yielded amorphene-type sesquiterpenes, indicating the co-occurrence of initial 1,6 and 1,10 cyclization of farnesyl diphosphate in a single enzyme. Interestingly, the immediate downstream oxidase CYP71AV1 had very low affinity to the side products of the recombinant ADS, including amorpha-4-en-7-ol, which is structurally similar to amorpha-4,11-diene. Our data uncover the complex catalytic mechanism of recombinant ADS and reveal a potential negative effect of the side products of recombinant ADS on the production of the artemisinin precursor in microbes.

Recent Advances and Future Perspectives in Cotton Research.

Cotton is not only the world's most important natural fiber crop, but it is also an ideal system in which to study genome evolution, polyploidization, and cell elongation. With the assembly of five different cotton genomes, a cotton-specific whole-genome duplication with an allopolyploidization process that combined the A- and D-genomes became evident. All existing A-genomes seemed to originate from the A0-genome as a common ancestor, and several transposable element bursts contributed to A-genome size expansion and speciation. The ethylene production pathway is shown to regulate fiber elongation. A tip-biased diffuse growth mode and several regulatory mechanisms, including plant hormones, transcription factors, and epigenetic modifications, are involved in fiber development. Finally, we describe the involvement of the gossypol biosynthetic pathway in the manipulation of herbivorous insects, the role of GoPGF in gland formation, and host-induced gene silencing for pest and disease control. These new genes, modules, and pathways will accelerate the genetic improvement of cotton.

Amorpha-4,11-diene synthase: a key enzyme in artemisinin biosynthesis and engineering.

Amorpha-4,11-diene synthase (ADS) catalyzes the first committed step in the artemisinin biosynthetic pathway, which is the first catalytic reaction enzymatically and genetically characterized in artemisinin biosynthesis. The advent of ADS in Artemisia annua is considered crucial for the emergence of the specialized artemisinin biosynthetic pathway in the species. Microbial production of amorpha-4,11-diene is a breakthrough in metabolic engineering and synthetic biology. Recently, numerous new techniques have been used in ADS engineering; for example, assessing the substrate promiscuity of ADS to chemoenzymatically produce artemisinin. In this review, we discuss the discovery and catalytic mechanism of ADS, its application in metabolic engineering and synthetic biology, as well as the role of sesquiterpene synthases in the evolutionary origin of artemisinin.

Genome-wide characterization of the GRF family and their roles in response to salt stress in Gossypium.

BACKGROUND: Cotton (Gossypium spp.) is the most important world-wide fiber crop but salt stress limits cotton production in coastal and other areas. Growth regulation factors (GRFs) play regulatory roles in response to salt stress, but their roles have not been studied in cotton under salt stress. RESULTS: We identified 19 GRF genes in G. raimondii, 18 in G. arboreum, 34 in G. hirsutum and 45 in G. barbadense, respectively. These GRF genes were phylogenetically analyzed leading to the recognition of seven GRF clades. GRF genes from diploid cottons (G. raimondii and G. arboreum) were largely retained in allopolyploid cotton, with subsequent gene expansion in G. barbadense relative to G. hirsutum. Most G. hirsutum GRF (GhGRF) genes are preferentially expressed in young and growing tissues. To explore their possible role in salt stress, we used qRT-PCR to study expression responses to NaCl treatment, showing that five GhGRF genes were down-regulated in leaves. RNA-seq experiments showed that seven GhGRF genes exhibited decreased expression in leaves under NaCl treatment, three of which (GhGRF3, GhGRF4, and GhGRF16) were identified by both RNA-seq and qRT-PCR. We also identified six and three GRF genes that exhibit decreased expression under salt stress in G. arboreum and G. barbadense, respectively. Consistent with its lack of leaf withering or yellowing under the salt treatment conditions, G. arboreum had better salt tolerance than G. hirsutum and G. barbadense. Our results suggest that GRF genes are involved in salt stress responses in Gossypium. CONCLUSION: In summary, we identified candidate GRF genes that were involved in salt stress responses in cotton.

RES transformation for biosynthesis and detoxification.

The reactive electrophilic species (RES), typically the molecules bearing α,ß-unsaturated carbonyl group, are widespread in living organisms and notoriously known for their damaging effects. Many of the mycotoxins released from phytopathogenic fungi are RES and their contamination to cereals threatens food safety worldwide. However, due to their high reactivity, RES are also used by host organisms to synthesize specific metabolites. The evolutionary conserved glyoxalase (GLX) system scavenges the cytotoxic α-oxoaldehydes that bear RES groups, which cause host disorders and diseases. In cotton, a specialized enzyme derived from glyoxalase I (GLXI) through gene duplications and named as specialized GLXI (SPG), acts as a distinct type of aromatase in the gossypol pathway to transform the RES intermediates into the phenolic products. In this review, we briefly introduce the research progress in understanding the RES, especially the RES-type mycotoxins, the GLX system and SPG, and discuss their application potential in detoxification and synthetic biology.

Aromatization of natural products by a specialized detoxification enzyme.

In plants, lineage-specific metabolites can be created by activities derived from the catalytic promiscuity of ancestral proteins, although examples of recruiting detoxification systems to biosynthetic pathways are scarce. The ubiquitous glyoxalase (GLX) system scavenges the cytotoxic methylglyoxal, in which GLXI isomerizes the α-hydroxy carbonyl in the methylglyoxal-glutathione adduct for subsequent hydrolysis. We show that GLXIs across kingdoms are more promiscuous than recognized previously and can act as aromatases without cofactors. In cotton, a specialized GLXI variant, SPG, has lost its GSH-binding sites and organelle-targeting signal, and evolved to aromatize cyclic sesquiterpenes bearing α-hydroxyketones to synthesize defense compounds in the cytosol. Notably, SPG is able to transform acetylated deoxynivalenol, the prevalent mycotoxin contaminating cereals and foods. We propose that detoxification enzymes are a valuable source of new catalytic functions and SPG, a standalone enzyme catalyzing complex reactions, has potential for toxin degradation, crop engineering and design of novel aromatics.

A gossypol biosynthetic intermediate disturbs plant defence response.

Plant secondary metabolites and their biosynthesis have attracted great interest, but investigations of the activities of hidden intermediates remain rare. Gossypol and related sesquiterpenes are the major phytoalexins in cotton. Among the six biosynthetic intermediates recently identified, 8-hydroxy-7-keto-δ-cadinene (C234) crippled the plant disease resistance when accumulated upon gene silencing. C234 harbours an α,ß-unsaturated carbonyl thus is a reactive electrophile species. Here, we show that C234 application also dampened the Arabidopsis resistance against the bacterial pathogen Pseudomonas syringae pv. maculicola ( Psm). We treated Arabidopsis with C234, Psm and ( Psm+C234), and analysed the leaf transcriptomes. While C234 alone exerted a mild effect, it greatly stimulated an over-response to the pathogen. Of the 7335 genes affected in the ( Psm+C234)-treated leaves, 3476 were unresponsive without the chemical, in which such functional categories as 'nucleotides transport', 'vesicle transport', 'MAP kinases', 'G-proteins', 'protein assembly and cofactor ligation' and 'light reaction' were enriched, suggesting that C234 disturbed certain physiological processes and the protein complex assembly, leading to distorted defence response and decreased disease resistance. As C234 is efficiently metabolized by CYP71BE79, plants of cotton lineage have evolved a highly active enzyme to prevent the phytotoxic intermediate accumulation during gossypol pathway evolution. This article is part of the theme issue 'Biotic signalling sheds light on smart pest management'.

Characterization of gossypol biosynthetic pathway.

Gossypol and related sesquiterpene aldehydes in cotton function as defense compounds but are antinutritional in cottonseed products. By transcriptome comparison and coexpression analyses, we identified 146 candidates linked to gossypol biosynthesis. Analysis of metabolites accumulated in plants subjected to virus-induced gene silencing (VIGS) led to the identification of four enzymes and their supposed substrates. In vitro enzymatic assay and reconstitution in tobacco leaves elucidated a series of oxidative reactions of the gossypol biosynthesis pathway. The four functionally characterized enzymes, together with (+)-δ-cadinene synthase and the P450 involved in 7-hydroxy-(+)-δ-cadinene formation, convert farnesyl diphosphate (FPP) to hemigossypol, with two gaps left that each involves aromatization. Of six intermediates identified from the VIGS-treated leaves, 8-hydroxy-7-keto-δ-cadinene exerted a deleterious effect in dampening plant disease resistance if accumulated. Notably, CYP71BE79, the enzyme responsible for converting this phytotoxic intermediate, exhibited the highest catalytic activity among the five enzymes of the pathway assayed. In addition, despite their dispersed distribution in the cotton genome, all of the enzyme genes identified show a tight correlation of expression. Our data suggest that the enzymatic steps in the gossypol pathway are highly coordinated to ensure efficient substrate conversion.

Systematic identification of functional residues of Artemisia annua amorpha-4,11-diene synthase.

Terpene synthases (TPSs) are responsible for the extremely diversified and complex structure of terpenoids. Amorpha-4,11-diene synthase (ADS) has a high (90%) fidelity in generating the sesquiterpene precursor for the biosynthesis of artemisinin, an antimalarial drug, however, little is known about how active site residues of ADS are involved in carbocation rearrangement and cyclization reactions. Here, we identify seven residues that are key to most of the catalytic steps in ADS. By structural modeling and amino acid sequence alignments of ADS with two functionally relevant sesquiterpene synthases from Artemisia annua, we performed site-directed mutagenesis and found that a single substitution, T296V, impaired the ring closure activity almost completely, and tetra-substitutions (L374Y/L404V/L405I/G439S) led to an enzyme generating 80% monocyclic bisabolyl-type sesquiterpenes, whereas a double mutant (T399L/T447G) showed compromised activity in regioselective deprotonation to yield 34.7 and 37.7% normal and aberrant deprotonation products, respectively. Notably, Thr296, Leu374, Gly439, Thr399, and Thr447, which play a major role in directing catalytic cascades, are located around conserved metal-binding motifs and function through impacting the folding of the substrate/intermediate, implying that residues surrounding the two motifs could be valuable targets for engineering TPS activity. Using this knowledge, we substantially increased amorpha-4,11-diene production in a near-additive manner by engineering Thr399 and Thr447 for product release. Our results provide new insight for the rational design of enzyme activity using synthetic biology.

Neural-network predictive control for nonlinear dynamic systems with time-delay.

A new recurrent neural-network predictive feedback control structure for a class of uncertain nonlinear dynamic time-delay systems in canonical form is developed and analyzed. The dynamic system has constant input and feedback time delays due to a communications channel. The proposed control structure consists of a linearized subsystem local to the controlled plant and a remote predictive controller located at the master command station. In the local linearized subsystem, a recurrent neural network with on-line weight tuning algorithm is employed to approximate the dynamics of the time-delay-free nonlinear plant. No linearity in the unknown parameters is required. No preliminary off-line weight learning is needed. The remote controller is a modified Smith predictor that provides prediction and maintains the desired tracking performance; an extra robustifying term is needed to guarantee stability. Rigorous stability proofs are given using Lyapunov analysis. The result is an adaptive neural net compensation scheme for unknown nonlinear systems with time delays. A simulation example is provided to demonstrate the effectiveness of the proposed control strategy.