Genome-wide identification of JAZ gene family in sugarcane and function analysis of ScJAZ1/2 in drought stress response and flowering regulation.

The JASMONATE ZIM DOMAIN (JAZ) proteins are a key inhibitors of the jasmonic acid (JA) signaling pathway that play an important role in the regulation of plant growth and development and environmental stress responses. However, there is no systematic identification and functional analysis of JAZ gene family members in sugarcane. In this study, a total of 49 SsJAZ genes were identified from the wild sugarcane species Saccharum spontaneum genome that were unevenly distributed on 13 chromosomes. Phylogenetic analysis showed that all SsJAZ members can be divided into six groups, and most of the SsJAZ genes contained photoreactive and ABA-responsive elements. RNA-seq analysis revealed that SsJAZ1-1/2/3/4 and SsJAZ7-1 were significantly upregulated under drought stress. The transcript level of ScJAZ1 which is the homologous gene of SsJAZ1 in modern sugarcane cultivars was upregulated by JA, PEG, and abscisic acid (ABA). Moreover, ScJAZ1 can interact with three other JAZ proteins to form heterodimers. The spatial and temporal expression analysis showed that SsJAZ2-1/2/3/4 were highly expressed in different tissues and growth stages and during the day-night rhythm between 10:00 and 18:00. Overexpression of ScJAZ2 in Arabidopsis accelerated flowering through activating the expression of AtSOC1, AtFT, and AtLFY. Moreover, the transcription level of ScJAZ2 was about 30-fold in the early-flowering sugarcane variety than that of the non-flowering variety, indicating ScJAZ2 positively regulated flowering. This first systematic analysis of the JAZ gene family and function analysis of ScJAZ1/2 in sugarcane provide key candidate genes and lay the foundation for sugarcane breeding.

Novel insights into the mechanism of laccase-driven rhizosphere humification for alleviating wheat 17ß-estradiol contamination.

Global-scale crop contamination with environmental estrogens has posed a huge risk to agri-food safety and human health. Laccase is regarded as an unexceptionable biocatalyst for regulating pollution and expediting humification, but the knowledge of estrogen bioremediation and C storage strengthened by laccase-driven rhizosphere humification (LDRH) remains largely unknown. Herein, a greenhouse microcosm was performed to explore the migration and fate of 17ß-estradiol (E2) in water-wheat (Triticum aestivum L.) matrices by LDRH. Compared to the non-added laccase, the pseudo-first-order decay rate constants of E2 in the rhizosphere solution after 10 and 50 µM exposures by LDRH increased from 0.03 and 0.02 h-1 to 0.36 and 0.09 h-1, respectively. Furthermore, LDRH conferred higher yield, polymerizability, O-containing groups, and functional-C signals in the humified precipitates, because it accelerated the formation of highly complex precipitates by radical-controlled continuous polymerization. In particular, not only did LDRH mitigate the phytotoxicity of E2, but it also diminished the metabolic load of E2 in wheat tissues. This was attributed to the rapid attenuation of E2 in the rhizosphere solution during LDRH, which limited E2 uptake and accumulation in each subcellular fraction of the wheat roots and shoots. Although several typical intermediate products such as estrone, estriol, and E2 oligomers were detected in roots, only small-molecule species were found in shoots, evidencing that the polymeric products of E2 were unable to be translocated acropetally due to the vast hydrophobicity and biounavailability. For the first time, our study highlights a novel, eco-friendly, and sustainable candidate for increasing the low-C treatment of organics in rhizosphere microenvironments and alleviating the potential risks of estrogenic contaminants in agroenvironments.

Genome-wide identification of GTE family proteins in sugarcane (Saccharum spontaneum) reveals that SsGTEL3a confers drought tolerance.

The bromodomain is a highly conserved protein domain that specifically binds to acetylated lysine residues in histones, thereby activating transcription of target genes. Although some progress in Global Transcription Factor Group E (GTE) has been achieved in numerous animals and a few plant species, no systematic analysis of GTE gene families has been reported yet in sugarcane. In our study, 37 GTE and GTE-Like (GTEL) genes were characterized in the Saccharum spontaneum. All SsGTE/SsGTEL members were heterogeneously located on all chromosomes of the sugarcane genome and divided into five groups. Transcriptome data showed that SsGTEL3a was expressed at significantly higher levels under drought stress in drought-resistant varieties than in drought-sensitive varieties. Moreover, the overexpression of SsGTEL3a significantly improved the drought tolerance in Arabidopsis through improving the scavenging ability of reactive oxygen species. Additionally, an interaction between ScFAR1 and SsGTEL3a was identified, with ScFAR1 showing a positive response to drought stress in bacterium. In summary, this systematic analysis of GTE gene family in sugarcane and functional research of SsGTEL3a broadened deeper insight into their evolutionary dynamics and functional properties and provided new candidate genes for drought-resistant molecular breeding of sugarcane.

Exolaccase Propels Humification to Decontaminate Bisphenol A and Create Humic-like Biostimulants.

Exolaccase-propelled humification (E-PH) helps eliminate phenolic pollutants and produce macromolecular precipitates. Herein, we investigated the influencing mechanism of 12 humic precursors (HPs) on exolaccase-enabled bisphenol A (BPA) decontamination and humification. Catechol, vanillic acid, caffeic acid, and gentian acid not only expedited BPA removal but also created large amounts of copolymeric precipitates. These precipitates had rich functional groups similar to natural humic substances, which presented great aromatic and acidic characteristics. The releasing amounts of BPA monomer from four precipitates were 0.08-12.87% at pH 2.0-11.0, suggesting that BPA-HP copolymers had pH stability. More excitingly, certain copolymeric precipitates could stimulate the growth and development of radish seedlings. The radish growth-promotion mechanisms of copolymers were involved in two aspects: (1) Copolymers interacted with root exudates to accelerate nutrient uptake; (2) Copolymers released auxins to provoke radish growth. These results may provide an innovative strategy for decontaminating phenolic pollutants and yielding humic-like biostimulants in E-PH.

GhWRKY41 forms a positive feedback regulation loop and increases cotton defence response against Verticillium dahliae by regulating phenylpropanoid metabolism.

Despite the established significance of WRKY proteins and phenylpropanoid metabolism in plant immunity, how WRKY proteins modulate aspects of the phenylpropanoid pathway remains undetermined. To understand better the role of WRKY proteins in plant defence, we identified a cotton (Gossypium hirsutum) protein, GhWRKY41, that is, universally and rapidly induced in three disease-resistant cotton cultivars following inoculation with the plant pathogenic fungus, Verticillium dahliae. We show that overexpression of GhWRKY41 in transgenic cotton and Arabidopsis enhances resistance to V. dahliae, while knock-down increases cotton more susceptibility to the fungus. GhWRKY41 physically interacts with itself and directly activates its own transcription. A genome-wide chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq), in combination with RNA sequencing (RNA-seq) analyses, revealed that 43.1% of GhWRKY41-binding genes were up-regulated in cotton upon inoculation with V. dahliae, including several phenylpropanoid metabolism master switches, receptor kinases, and disease resistance-related proteins. We also show that GhWRKY41 homodimer directly activates the expression of GhC4H and Gh4CL, thereby modulating the accumulation of lignin and flavonoids. This finding expands our understanding of WRKY-WRKY protein interactions and provides important insights into the regulation of the phenylpropanoid pathway in plant immune responses by a WRKY protein.

Field evaluation of TaDREB2B-ectopic expression sugarcane (Saccharum spp. hybrid) for drought tolerance.

Sugarcane is one of the most crucial sugar crops globally that supplies the main raw material for sugar and ethanol production, but drought stress causes a severe decline in sugarcane yield worldwide. Enhancing sugarcane drought resistance and reducing yield and quality losses is an ongoing challenge in sugarcane genetic improvement. Here, we introduced a Tripidium arundinaceum dehydration-responsive element-binding transcription factor (TaDREB2B) behind the drought-responsible RD29A promoter into a commercial sugarcane cultivar FN95-1702 and subsequently conducted a series of drought tolerance experiments and investigation of agronomic and quality traits. Physiological analysis indicated that Prd29A: TaDREB2B transgenic sugarcane significantly confers drought tolerance in both the greenhouses and the field by enhancing water retention capacity and reducing membrane damage without compromising growth. These transgenic plants exhibit obvious improvements in yield performance and various physiological traits under the limited-irrigation condition in the field, such as increasing 41.9% yield and 44.4% the number of ratooning sugarcane seedlings. Moreover, Prd29A: TaDREB2B transgenic plants do not penalize major quality traits, including sucrose content, gravity purity, Brix, etc. Collectively, our results demonstrated that the Prd29A-TaDREB2B promoter-transgene combination will be a useful biotechnological tool for the increase of drought tolerance and the minimum of yield losses in sugarcane.

A Single-Nucleotide Mutation in a GLUTAMATE RECEPTOR-LIKE Gene Confers Resistance to Fusarium Wilt in Gossypium hirsutum.

Fusarium wilt (FW) disease of cotton, caused by the fungus Fusarium oxysporum f. sp. vasinfectum (Fov), causes severe losses in cotton production worldwide. Though significant advancements have been made in development of FW-resistant Upland cotton (Gossypium hirsutum) in resistance screening programs, the precise resistance genes and the corresponding molecular mechanisms for resistance to Fov remain unclear. Herein it is reported that Fov7, a gene unlike canonical plant disease-resistance (R) genes, putatively encoding a GLUTAMATE RECEPTOR-LIKE (GLR) protein, confers resistance to Fov race 7 in Upland cotton. A single nucleotide polymorphism (SNP) (C/A) in GhGLR4.8, resulting in an amino acid change (L/I), is associated with Fov resistance. A PCR-based DNA marker (GhGLR4.8SNP(A/C) ) is developed and shown to cosegregate with the Fov resistance. CRISPR/Cas9-mediated knockout of Fov7 results in cotton lines extremely susceptible to Fov race 7 with a loss of the ability to induce calcium influx in response to total secreted proteins (SEPs) of Fov. Furthermore, coinfiltration of SEPs with GhGLR4.8A results in a hypersensitive response. This first report of a GLR-encoding gene that functions as an R gene provides a new insight into plant-pathogen interactions and a new handle to develop cotton cultivars with resistance to Fov race 7.

Orchestration of plant development and defense by indirect crosstalk of salicylic acid and brassinosteorid signaling via transcription factor GhTINY2.

Salicylic acid (SA) and brassinosteroids (BRs) are well known to regulate diverse processes of plant development and stress responses, but the mechanisms by which these phytohormones mediate the growth and defense trade-off are largely unclear. In addition, little is known about the roles of DEHYDRATION RESPONSIVE ELEMENT BINDING transcription factors, especially in biotic stress and plant growth. Here, we identified a cotton (Gossypium hirsutum) APETALA2/ETHYLENE RESPONSIVE FACTOR gene GhTINY2 that is strongly induced by Verticillium dahliae. Overexpression of GhTINY2 in cotton and Arabidopsis enhanced tolerance to V. dahliae, while knockdown of expression increased the susceptibility of cotton to the pathogen. GhTINY2 was found to promote SA accumulation and SA signaling transduction by directly activating expression of WRKY51. Moreover, GhTINY2-overexpressing cotton and Arabidopsis showed retardation of growth, increased sensitivity to inhibitors of BR biosynthesis, down-regulation of several BR-induced genes, and up-regulation of BR-repressed genes, while GhTINY2-RNAi cotton showed the opposite effects. We further determined that GhTINY2 negatively regulates BR signaling by interacting with BRASSINAZOLE-RESISTANT 1 (BZR1) and restraining its transcriptional activation of the expression of INDOLE-3-ACETIC ACID INDUCIBLE 19 (IAA19). These findings indicate that GhTINY2 fine-tunes the trade-off between immunity and growth via indirect crosstalk between WRKY51-mediated SA biosynthesis and BZR1-IAA19-regulated BR signaling.

GhWRKY1-like enhances cotton resistance to Verticillium dahliae via an increase in defense-induced lignification and S monolignol content.

Cotton is one of the most important economic crops and is cultivated globally. Verticillium wilt, caused by the soil-borne hemibiotrophic fungus Verticillium dahliae, is the most destructive disease in cotton production for its infection strategies and great genetic plasticity. Recent studies have identified the accumulation of lignin is a general and basal defense reaction in plant immunity and cotton resistance to V. dahliae. However, the functions and regulatory mechanisms of transcription factors in cotton defense-induced lignification and lignin composition alteration were less reported. Here, we identified a WRKY transcription factor GhWRKY1-like from upland cotton (Gossypium hirsutum) as a positive regulator in resistance to V. dahliae via directly manipulating lignin biosynthesis. Further analysis revealed that GhWRKY1-like interacts with the promoters of lignin biosynthesis related genes GhPAL6 and GhCOMT1, and activates the expression of GhPAL6 and GhCOMT1, which led to enhanced total lignin especially S monomers biosynthesis. These results demonstrate that GhWRKY1-like enhances Verticillium wilt resistance via an increase in defense-induced lignification and broaden our knowledge of the roles of lignification and the lignin composition in plant defense responses.

GhMYB4 downregulates lignin biosynthesis and enhances cotton resistance to Verticillium dahliae.

KEY MESSAGE: GhMYB4 acts as a negative regulator in lignin biosynthesis, which results in alteration of cell wall integrity and activation of cotton defense response. Verticillium wilt of cotton (Gossypium hirsutum) caused by the soil-borne fungus Verticillium dahliae (V. dahliae) represents one of the most important constraints of cotton production worldwide. Mining of the genes involved in disease resistance and illuminating the molecular mechanisms that underlie this resistance is of great importance in cotton breeding programs. Defense-induced lignification in plants is necessary for innate immunity, and there are reports of a correlation between increased lignification and disease resistance. In this study, we present an example in cotton whereby plants with reduced lignin content also exhibit enhanced disease resistance. We identified a negative regulator of lignin synthesis, in cotton encoded in GhMYB4. Overexpression of GhMYB4 in cotton and Arabidopsis enhanced resistance to V. dahliae  with reduced lignin deposition. Moreover, GhMYB4 could bind the promoters of several genes involved in lignin synthesis, such as GhC4H-1, GhC4H-2, Gh4CL-4, and GhCAD-3, and impair their expression. The reduction of lignin content in GhMYB4-overexpressing cotton led to alterations of cell wall integrity (CWI) and released more oligogalacturonides (OGs) which may act as damage-associated molecular patterns (DAMPs) to stimulate plant defense responses. In support of this hypothesis, exogenous application with polygalacturonic acid (PGA) in cotton activated biosynthesis of jasmonic acid (JA) and JA-mediated defense against V. dahliae, similar to that described for cotton plants overexpressing GhMYB4. This study provides a new candidate gene for cotton disease-resistant breeding and an increased understanding of the relationship between lignin synthesis, OG release, and plant immunity.

Phosphate deficiency enhances cotton resistance to Verticillium dahliae through activating jasmonic acid biosynthesis and phenylpropanoid pathway.

Living in natural environment, plants often suffer from various biotic and abiotic stresses. Phosphate deficiency is a common factor affecting crop production in field, while pathogen invasion is another serious problem. Here we report that Pi-deficient cotton plants exhibit enhanced resistance to Verticillium dahliae. Transcriptomic and histochemical analysis revealed that cotton phenylpropanoid pathway was activated under phosphate deficiency, including lignin and flavonoid biosynthesis. Metabolomic data showed that Pi-deficient cotton accumulates many flavonoids metabolites and displays obvious anti-fungi activity in terms of methanolic extract. Additionally, JA biosynthesis was activated under phosphate deficiency and the Pi-deficiency induced disease resistance was significantly attenuated in GhAOS knock down plants. Taken together, our study demonstrated that phosphate deficiency enhanced cotton resistance to V. dahliae through activating phenylpropanoid pathway and JA biosynthesis, providing new insights into how phosphate deficiency affects plant disease resistance.

GhCPK33 Negatively Regulates Defense against Verticillium dahliae by Phosphorylating GhOPR3.

Verticillium wilt, caused by the soil-borne fungus Verticillium dahliae, is a destructive vascular disease in plants. Approximately 200 dicotyledonous plant species in temperate and subtropical regions are susceptible to this notorious pathogen. Previous studies showed that jasmonic acid (JA) plays a crucial role in plant-V. dahliae interactions. V. dahliae infection generally induces significant JA accumulation in local and distal tissues of the plant. Although JA biosynthesis and the associated enzymes have been studied intensively, the precise mechanism regulating JA biosynthesis upon V. dahliae infection remains unknown. Here, we identified the calcium-dependent protein kinase GhCPK33 from upland cotton (Gossypium hirsutum) as a negative regulator of resistance to V. dahliae that directly manipulates JA biosynthesis. Knockdown of GhCPK33 by Agrobacterium tumefaciens-mediated virus-induced gene silencing constitutively activated JA biosynthesis and JA-mediated defense responses and enhanced resistance to V dahliae Further analysis revealed that GhCPK33 interacts with 12-oxophytodienoate reductase3 (GhOPR3) in peroxisomes. GhCPK33 phosphorylates GhOPR3 at threonine-246, leading to decreased stability of GhOPR3, which consequently limits JA biosynthesis. We propose that GhCPK33 is a potential molecular target for improving resistance to Verticillium wilt disease in cotton.