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.

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.

The sage genome provides insight into the evolutionary dynamics of diterpene biosynthesis gene cluster in plants.

The widely cultivated medicinal and ornamental plant sage (Salvia officinalis L.) is an evergreen shrub of the Lamiaceae family, native to the Mediterranean. We assembled a high-quality sage genome of 480 Mb on seven chromosomes, and identified a biosynthetic gene cluster (BGC) encoding two pairs of diterpene synthases (diTPSs) that, together with the cytochromes P450 (CYPs) genes located inside and outside the cluster, form two expression cascades responsible for the shoot and root diterpenoids, respectively, thus extending BGC functionality from co-regulation to orchestrating metabolite production in different organs. Phylogenomic analysis indicates that the Salvia clades diverged in the early Miocene. In East Asia, most Salvia species are herbaceous and accumulate diterpenoids in storage roots. Notably, in Chinese sage S. miltiorrhiza, the diterpene BGC has contracted and the shoot cascade has been lost. Our data provide genomic insights of micro-evolution of growth type-associated patterning of specialized metabolite production in plants.

Sphingolipid metabolism, transport, and functions in plants: Recent progress and future perspectives.

Sphingolipids, which comprise membrane systems together with other lipids, are ubiquitous in cellular organisms. They show a high degree of diversity across plant species and vary in their structures, properties, and functions. Benefiting from the development of lipidomic techniques, over 300 plant sphingolipids have been identified. Generally divided into free long-chain bases (LCBs), ceramides, glycosylceramides (GlcCers) and glycosyl inositol phosphoceramides (GIPCs), plant sphingolipids exhibit organized aggregation within lipid membranes to form raft domains with sterols. Accumulating evidence has revealed that sphingolipids obey certain trafficking and distribution rules and confer unique properties to membranes. Functional studies using sphingolipid biosynthetic mutants demonstrate that sphingolipids participate in plant developmental regulation, stimulus sensing, and stress responses. Here, we present an updated metabolism/degradation map and summarize the structures of plant sphingolipids, review recent progress in understanding the functions of sphingolipids in plant development and stress responses, and review sphingolipid distribution and trafficking in plant cells. We also highlight some important challenges and issues that we may face during the process of studying sphingolipids.

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.

Mediation of JA signalling in glandular trichomes by the woolly/SlMYC1 regulatory module improves pest resistance in tomato.

Almost all plants form trichomes, which protect them against insect herbivores by forming a physical barrier and releasing chemical repellents. Glandular trichomes produce a variety of specialized defensive metabolites, including volatile terpenes. Previous studies have shown that the defence hormone jasmonic acid (JA) affects trichome development and induces terpene synthases (TPSs) but the underlying molecular mechanisms remain unclear. Here, we characterized a loss-of-function allele of the HD-ZIP IV transcription factor woolly (wo) and analysed its role in mediating JA signalling in tomato. We showed that knockout of wo led to extensive trichome defects, including structural and functional changes in type VI glandular trichomes, and a dramatic reduction in terpene levels. We further found that wo directly binds to TPS gene promoters to recruit SlMYC1, a JA signalling modulator, and that together these transcription factors promote terpene biosynthesis in tomato trichomes. The wo/SlMYC1 regulatory module is inhibited by SlJAZ2 through a competitive binding mechanism, resulting in a fine-tuned JA response in tomato trichomes. Enhanced expression of SlMYC1 substantially increased terpene levels and improved tomato resistance to spider mites. Interestingly, we also found that SlMYC1 plays an additional role in glandular cell division and expansion in type VI trichomes, independent of JA. Together, our results reveal a novel, JA-mediated regulatory mechanism that promotes insect resistance in tomato.

Arabidopsis leaf extracellular vesicles in wound-induced jasmonate accumulation.

The plant extracellular vesicles (EVs) are lipid-enveloped nano-particles containing proteins, nucleic acids and metabolites and function in plant development and response. The Arabidopsis four transmembrane protein TETRASPANIN 8 (TET8) knock-out mutant tet8 secreted less EVs than the wild-type (WT). In this report, we show that the tet8 mutant was attenuated in the plant hormone jasmonate (JA) accumulation in response to mechanical wounding treatment. We also noticed that the EVs contained a high level of phospholipids phosphatidic acids (PAs) which may serve as precursors of JA biosynthesis during wound-triggered-self-healing processes. Thus, we propose an open question about a potential role of EVs or TET8 or both in damage-associated JA response.

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.

Lipidomic Analysis Reveals the Importance of GIPCs in Arabidopsis Leaf Extracellular Vesicles.

Plant extracellular vesicles (EVs) are membrane-enclosed nanoparticles that play diverse roles in plant development and response. Recently, impressive progress has been made in the isolation and identification of the proteins and RNAs carried in plant EVs; however, the analysis of EV lipid compositions remains rudimentary. Here, we performed lipidomic analysis of Arabidopsis rosette leaf EVs, revealing a high abundance of certain groups of lipids, in particular sphingolipids, in the EVs. Remarkably, the EV sphingolipids are composed of nearly pure glycosylinositolphosphoceramides (GIPCs), which are green lineage abundant and negatively charged. We further showed that the Arabidopsis TETRASPANIN 8 (TET8) knockout mutant has a lower amount of cellular GIPCs and secrets fewer EVs, companied with impaired reactive oxygen species (ROS) burst toward stresses. Exogenous application of GIPCs promoted the secretion of EVs and ROS burst in both the WT and tet8 mutant. The characteristic enrichment of sphingolipid GIPCs provides valuable insights into the biogenesis and function of plant EVs.

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.

The miR319-Targeted GhTCP4 Promotes the Transition from Cell Elongation to Wall Thickening in Cotton Fiber.

Plant cell growth involves a complex interplay among cell-wall expansion, biosynthesis, and, in specific tissues, secondary cell wall (SCW) deposition, yet the coordination of these processes remains elusive. Cotton fiber cells are developmentally synchronous, highly elongated, and contain nearly pure cellulose when mature. Here, we report that the transcription factor GhTCP4 plays an important role in balancing cotton fiber cell elongation and wall synthesis. During fiber development the expression of miR319 declines while GhTCP4 transcript levels increase, with high levels of the latter promoting SCW deposition. GhTCP4 interacts with a homeobox-containing factor, GhHOX3, and repressing its transcriptional activity. GhTCP4 and GhHOX3 function antagonistically to regulate cell elongation, thereby establishing temporal control of fiber cell transition to the SCW stage. We found that overexpression of GhTCP4A upregulated and accelerated activation of the SCW biosynthetic pathway in fiber cells, as revealed by transcriptome and promoter activity analyses, resulting in shorter fibers with varied lengths and thicker walls. In contrast, GhTCP4 downregulation led to slightly longer fibers and thinner cell walls. The GhHOX3-GhTCP4 complex may represent a general mechanism of cellular development in plants since both are conserved factors in many species, thus providing us a potential molecular tool for the design of fiber traits.

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.

Core cis-element variation confers subgenome-biased expression of a transcription factor that functions in cotton fiber elongation.

Cotton cultivars have evolved to produce extensive, long, seed-born fibers important for the textile industry, but we know little about the molecular mechanism underlying spinnable fiber formation. Here, we report how PACLOBUTRAZOL RESISTANCE 1 (PRE1) in cotton, which encodes a basic helix-loop-helix (bHLH) transcription factor, is a target gene of spinnable fiber evolution. Differential expression of homoeologous genes in polyploids is thought to be important to plant adaptation and novel phenotypes. PRE1 expression is specific to cotton fiber cells, upregulated during their rapid elongation stage and A-homoeologous biased in allotetraploid cultivars. Transgenic studies demonstrated that PRE1 is a positive regulator of fiber elongation. We determined that the natural variation of the canonical TATA-box, a regulatory element commonly found in many eukaryotic core promoters, is necessary for subgenome-biased PRE1 expression, representing a mechanism underlying the selection of homoeologous genes. Thus, variations in the promoter of the cell elongation regulator gene PRE1 have contributed to spinnable fiber formation in cotton. Overexpression of GhPRE1 in transgenic cotton yields longer fibers with improved quality parameters, indicating that this bHLH gene is useful for improving cotton fiber quality.

Jasmonate response decay and defense metabolite accumulation contributes to age-regulated dynamics of plant insect resistance.

Immunity deteriorates with age in animals but comparatively little is known about the temporal regulation of plant resistance to herbivores. The phytohormone jasmonate (JA) is a key regulator of plant insect defense. Here, we show that the JA response decays progressively in Arabidopsis. We show that this decay is regulated by the miR156-targeted SQUAMOSA PROMOTER BINDING PROTEIN-LIKE9 (SPL9) group of proteins, which can interact with JA ZIM-domain (JAZ) proteins, including JAZ3. As SPL9 levels gradually increase, JAZ3 accumulates and the JA response is attenuated. We provide evidence that this pathway contributes to insect resistance in young plants. Interestingly however, despite the decay in JA response, older plants are still comparatively more resistant to both the lepidopteran generalist Helicoverpa armigera and the specialist Plutella xylostella, along with increased accumulation of glucosinolates. We propose a model whereby constitutive accumulation of defense compounds plays a role in compensating for age-related JA-response attenuation during plant maturation.

Isolation and Characterization of Three New Monoterpene Synthases from Artemisia annua.

Artemisia annua, an annual herb used in traditional Chinese medicine, produces a wealth of monoterpenes and sesquiterpenes, including the well-known sesquiterpene lactone artemisinin, an active ingredient in the treatment for malaria. Here we report three new monoterpene synthases of A. annua. From a glandular trichome cDNA library, monoterpene synthases of AaTPS2, AaTPS5, and AaTPS6, were isolated and characterized. The recombinant proteins of AaTPS5 and AaTPS6 produced multiple products with camphene and 1,8-cineole as major products, respectively, and AaTPS2 produced a single product, ß-myrcene. Although both Mg(2+) and Mn(2+) were able to support their catalytic activities, altered product spectrum was observed in the presence of Mn(2+) for AaTPS2 and AaTPS5. Analysis of extracts of aerial tissues and root of A. annua with gas chromatography-mass spectrometry detected more than 20 monoterpenes, of which the three enzymes constituted more than 1/3 of the total. Mechanical wounding induced the expression of all three monoterpene synthase genes, and transcript levels of AaTPS5 and AaTPS6 were also elevated after treatments with phytohormones of methyl jasmonate, salicylic acid, and gibberellin, suggesting a role of these monoterpene synthases in plant-environment interactions. The three new monoterpene synthases reported here further our understanding of molecular basis of monoterpene biosynthesis and regulation in plant.

Functional characterization of a basic helix-loop-helix (bHLH) transcription factor GhDEL65 from cotton (Gossypium hirsutum).

Cotton fiber is proposed to share some similarity with the Arabidopsis thaliana leaf trichome, which is regulated by the MYB-bHLH-WD40 transcription complex. Although several MYB transcription factors and WD40 family proteins in cotton have been characterized, little is known about the role of bHLH family proteins in cotton. Here, we report that GhDEL65, a bHLH protein from cotton (Gossypium hirsutum), is a functional homologue of Arabidopsis GLABRA3 (GL3) and ENHANCER OF GLABRA3 (EGL3) in regulating trichome development. Transcripts of GhDEL65 were detected in 0 ∼ 1 days post-anthesis (DPA) ovules and abundant in 3-DPA fibers, implying that GhDEL65 may act in early fiber development. Ectopic expression of GhDEL65 in Arabidopsis gl3 egl3 double mutant partly rescued the trichome development, and constitutive expression of GhDEL65 in wild-type plants led to increased trichome density on rosette leaves and stems, mainly by activating the transcription of two key positive regulators of trichome development, GLABRA1 (GL1) and GLABRA2 (GL2), and suppressed the expression of a R3 single-repeat MYB factor TRIPTYCHON (TRY). GhDEL65 could interact with cotton R2R3 MYB transcription factors GhMYB2 and GhMYB3, as well as the WD40 protein GhTTG3, suggesting that the MYB-bHLH-WD40 protein complex also exists in cotton fiber cell, though its function in cotton fiber development awaits further investigation.