Identification of host gene regulation and resistance pathway dynamics at diverse infection stages of Rhizoctonia solani AG3-TB.

Rhizoctonia solani is a fungal pathogen that causes significant losses in agricultural production. Because of its rapid transmission and broad host range, the exploration of genes involved in defense responses to the infection of R. solani has become an important task. Here, we performed a time-course RNA-Seq experiment to explore crucial genes or pathways involved in host responses to R. solani AG3-TB infection at 6, 12, 24, 36, 48, and 72 hours post inoculation (hpi). GO and KEGG enrichment analysis revealed that most DEGs were enriched in the basal metabolism pathways, including carbohydrate metabolic processes and the biosynthesis of amino acids. Moreover, catalase (CAT) and superoxide dismutase (SOD) were up-regulated, and transcription factors (TFs) such as WRKY, AP2, and MYB were increased significantly compared to the control (0 hpi). Silencing of WRKY70 and catalase-3 exhibited elevated susceptibility to the fungal infection. To summarize, the TFs WRKY70 and WRKY75, genes involved in jasmonic acid (JA), salicylic acid (SA), and brassinosteroids (BR) signaling pathways, and defense-related enzymes may play crucial roles in the host responses to R. solani AG3-TB infection.

Glucanases and Chitinases in Mangifera indica: Identification, Classification, Phylogeny, and Expression Analysis of Defense Genes against Colletotrichum spp.

Plant glucanases and chitinases are defense proteins that participate in pathogenesis; however, very little is known about the glucanase (GLUC) and chitinase (CHIT) gene families in mango. Some mango cultivars are of great economic importance and can be affected by anthracnose, a postharvest disease caused by fungi of the genus Colletotrichum spp. This study identified and characterized 23 putative glucanases and 16 chitinases in the mango genome cv. Tommy Atkins. We used phylogenetic analyses to classify the glucanases into three subclasses (A, B, and C) and the chitinases into four classes (I, II, IV, and V). Information on the salicylic, jasmonic acid, and ethylene pathways was obtained by analyzing the cis-elements of the GLUC and CHIT class I and IV gene promoters. The expression profile of GLUC, CHIT class I, and CHIT class IV genes in mango cv. Ataulfo inoculated with two Colletotrichum spp. revealed different profile expression related to these fungi's level of virulence. In general, this study provides the basis for the functional validation of these target genes with which the regulatory mechanisms used by glucanases and chitinases as defense proteins in mango can be elucidated.

Gossypium arboreum PPD2 facilitates root architecture development to increase plant resilience to salt stress.

The jasmonic acid (JA) signaling pathway plays an important role in plant responses to abiotic stresses. The PEAPOD (PPD) and jasmonate ZIM-domain (JAZ) protein in the JA signaling pathway belong to the same family, but their functions in regulating plant defense against salt stress remain to be elucidated. Here, Gossypium arboreum PPD2 was overexpressed in Arabidopsis thaliana and systematically silenced in cotton for exploring its function in regulating plant defense to salt stress. The GaPPD2-overexpressed Arabidopsis thaliana plants significantly increased the tolerance to salt stress compared to the wild type in both medium and soil, while the GaPPD2-silenced cotton plants showed higher sensitivity to salt stress than the control in pots. The antioxidant activities experiment showed that GaPPD2 may mitigate the accumulation of reactive oxygen species by promoting superoxide dismutase accumulation, consequently improving plant resilience to salt stress. Through the exogenous application of MeJA (methy jasmonate) and the protein degradation inhibitor MG132, it was found that GaPPD2 functions in plant defense against salt stress and is involved in the JA signaling pathway. The RNA-seq analysis of GaPPD2-overexpressed A. thaliana plants and receptor materials showed that the differentially expressed genes were mainly enriched in antioxidant activity, peroxidase activity, and plant hormone signaling pathways. qRT-PCR results demonstrated that GaPPD2 might positively regulate plant defense by inhibiting GH3.2/3.10/3.12 expression and activating JAZ7/8 expression. The findings highlight the potential of GaPPD2 as a JA signaling component gene for improving the cotton plant resistance to salt stress and provide insights into the mechanisms underlying plant responses to environmental stresses.

Phase 1 study of NEDD8 activating enzyme inhibitor pevonedistat in combination with chemotherapy in pediatric patients with recurrent or refractory solid tumors (ADVL1615).

PURPOSE: The objective of this study was to determine the recommended Phase 2 dose (RP2D) of pevonedistat, a first in class inhibitor of NEDD8 activating enzyme, in combination with irinotecan (IRN) and temozolomide (TMZ) in children with cancer. METHODS: This Phase 1 study used a rolling 6 design to evaluate escalating doses of pevonedistat in combination with standard doses of IRN and TMZ in pediatric patients with recurrent/refractory solid or CNS tumors. During cycle 1, pevonedistat was administered intravenously on days 1, 8, 10, and 12, with IRN (IV, 50 mg/m2) and TMZ (orally, 100 mg/m2), on days 8-12 of a 28-day cycle. In subsequent cycles, pevonedistat was administered on days 1, 3, and 5, with IRN/TMZ on days 1-5 of a 21-day cycle. RESULTS: Thirty patients enrolled; all were eligible and evaluable for toxicity. Six patients each enrolled on pevonedistat dose levels (DL) 1 (15 mg/m2), 2 (20 mg/m2), 3 (25 mg/m2) and 4 (35 mg/m2) as well as an expanded pharmacokinetic (PK) cohort at DL4. The maximum tolerated dose (MTD) was not exceeded. 2/12 (17 %) patients treated at the RP2D (35 mg/m2) experienced a cycle 1 dose limiting toxicity (DLT). IRN is unlikely to affect the pharmacokinetics of pevonedistat. Two patients had a partial response and 6 patients had prolonged stable disease (> 6 cycles). CONCLUSIONS: Pevonedistat in combination with IRN/TMZ is well tolerated in children with solid or CNS tumors. The RP2D of pevonedistat is 35 mg/m2 on days 1, 3, 5 in combination with IRN/TMZ.

Molecular Changes during Germination of Cocoa Beans, Part 1.

Some germination is known to occur during the process of fermentation in cocoa beans. The impact of this biological process on the course of cocoa fermentation is not known and was thus investigated. In order to determine the impact of germination at the molecular level as well as on flavor, an untargeted metabolomics approach using Ultra Performance Liquid Chromatography-Electrospray Ionization-Time of Flight-Mass Spectrometry (UPLC-ESI-ToF-MS) with simultaneous acquisition of low- and high-collision energy mass spectra (MSe) was performed. Extracts of raw and germinated cocoa beans of the same origin were measured and compared for characteristic differences by unsupervised principal component analysis. OPLS-DA revealed 12-hydroxyjasmonic acid (HOJA) sulfate, (+)-catechin and (-)-epicatechin as most down-regulated compounds as well as two hydroxymethylglutaryl (HMG) glucosides A and B among others as decisive up-regulated compounds in the germinated material. Additionally, further HMG glucosides and 12-hydroxyjasmonic acid could be identified in cocoa for the first time by coelution with isolated and synthesized reference compounds. HOJA sulfate, which has been postulated in cocoa, and HOJA were revealed to impart bitter and astringent taste qualities.

NAD+ deficiency primes defense metabolism via 1O2-escalated jasmonate biosynthesis in plants.

Nicotinamide adenine dinucleotide (NAD+) is a redox cofactor and signal central to cell metabolisms. Disrupting NAD homeostasis in plant alters growth and stress resistance, yet the underlying mechanisms remain largely unknown. Here, by combining genetics with multi-omics, we discover that NAD+ deficiency in qs-2 caused by mutation in NAD+ biosynthesis gene-Quinolinate Synthase retards growth but induces biosynthesis of defense compounds, notably aliphatic glucosinolates that confer insect resistance. The elevated defense in qs-2 is resulted from activated jasmonate biosynthesis, critically hydroperoxidation of α-linolenic acid by the 13-lipoxygenase (namely LOX2), which is escalated via the burst of chloroplastic ROS-singlet oxygen (1O2). The NAD+ deficiency-mediated JA induction and defense priming sequence in plants is recapitulated upon insect infestation, suggesting such defense mechanism operates in plant stress response. Hence, NAD homeostasis is a pivotal metabolic checkpoint that may be manipulated to navigate plant growth and defense metabolism for stress acclimation.

Spider mite infestation triggers coordinated hormonal trade-offs enabling plant survival with a fitness cost.

Tetranychus urticae is an important pest that causes severe damage to a wide variety of plants and crops, leading to a substantial productivity loss. Previous research has been focused on plant defence response to T. urticae to improve plant resistance. However, plant growth, development and reproduction throughout the infestation process have not been previously studied. Through physiological, biochemical, transcriptomic and hormonomic evaluation, we uncover the molecular mechanisms directing the defence-growth trade-off established in Arabidopsis upon T. urticae infestation. Upon mite attack, plants suffer an adaptation process characterized by a temporal separation between the defence and growth responses. Jasmonic and salicylic acids regulate the main defence responses in combination with auxin and abscisic acid. However, while the reduction of both auxin signalling and gibberellin, cytokinin and brassinosteroid biosynthesis lead to initial growth arrest, increasing levels of growth hormones at later stages enables growth restart. These alterations lead to a plant developmental delay that impacts both seed production and longevity. We demonstrate that coordinated trade-offs determine plant adaptation and survival, revealing mite infestation has a long-lasting effect negatively impacting seed viability. This study provides additional tools to design pest management strategies that improve resistance without penalty in plant fitness.

Low concentrations of methyl jasmonate promote plant growth and mitigate Cd toxicity in Cosmos bipinnatus.

Cadmium (Cd) is a biologically non-essential heavy metal, a major soil pollutant, and extremely harmful to plants. The phytohormone methyl jasmonate (MeJA) plays an important role in plant heavy-metal resistance. However, the understanding of the effects of MeJA supply level on alleviating Cd toxicity in plants is limited. Here, we investigated how MeJA regulated the development of physiological processes and cell wall modification in Cosmos bipinnatus. We found that low concentrations of MeJA increased the dry weight of seedlings under 120 µM Cd stress by reducing the transport of Cd from roots to shoots. Moreover, a threshold concentration of exogenous MeJA increased the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) in plant roots, the concentration of Cd in the root cell wall, and the contents of pectin and hemicellulose 1 polysaccharides, through converting Cd into pectin-bound forms. These results suggested that MeJA mitigated Cd toxicity by modulating root cell wall polysaccharide and functional group composition, especially through pectin polysaccharides binding to Cd, with effects on Cd transport capacity, specific chemical forms of Cd, and homeostatic antioxidant systems in C. bipinnatus.

Terrein Exhibits Anti-tumor Activity by Suppressing Angiogenin Expression in Malignant Melanoma Cells.

BACKGROUND/AIM: Malignant melanoma is a tumor with a poor prognosis that can metastasize distally at an early stage. Terrein, a metabolite produced by Aspergillus terreus, suppresses the expression of angiogenin, an angiogenic factor. However, the pharmacological effects of natural terrein have not been elucidated, because only a small amount of terrein can be extracted from large fungal cultures. In this study, we investigated the antineoplastic effects of terrein on human malignant melanoma cells and its underlying mechanisms. MATERIALS AND METHODS: Human malignant melanoma cell lines were cultured in the presence of terrein and analyzed. Angiogenin production was evaluated using ELISA. Ribosome biosynthesis was evaluated using silver staining of the nucleolar organizer region. Intracellular signaling pathways were analyzed using western blotting. Malignant melanoma cells were transplanted subcutaneously into the backs of nude mice. The tumors were removed at 5 weeks and analyzed histopathologically. RESULTS: Terrein inhibited angiogenin expression, proliferation, migration, invasion, and ribosome biosynthesis in malignant melanoma cells. Terrein was shown to inhibit tumor growth and angiogenesis in animal models. CONCLUSION: This study demonstrated that terrein has anti-tumor effects against malignant melanoma. Furthermore, chemically synthesized non-natural terrein can be mass-produced and serve as a novel potential anti-tumor drug candidate.

Exploring the effects of red light night break on the defence mechanisms of tomato against fungal pathogen Botrytis cinerea.

Plant infections caused by fungi lead to significant crop losses worldwide every year. This study aims to better understand the plant defence mechanisms regulated by red light, in particular, the effects of red light at night when most phytopathogens are highly infectious. Our results showed that superoxide production significantly increased immediately after red light exposure and, together with hydrogen peroxide levels, was highest at dawn after 30 min of nocturnal red-light treatment. In parallel, red-light-induced expression and increased the activities of several antioxidant enzymes. The nocturnal red light did not affect salicylic acid but increased jasmonic acid levels immediately after illumination, whereas abscisic acid levels increased 3 h after nocturnal red-light exposure at dawn. Based on the RNAseq data, red light immediately increased the transcription of several chloroplastic chlorophyll a-b binding protein and circadian rhythm-related genes, such as Constans 1, CONSTANS interacting protein 1 and zinc finger protein CONSTANS-LIKE 10. In addition, the levels of several transcription factors were also increased after red light exposure, such as the DOF zinc finger protein and a MYB transcription factor involved in the regulation of circadian rhythms and defence responses in tomato. In addition to identifying these key transcription factors in tomato, the application of red light at night for one week not only reactivated key antioxidant enzymes at the gene and enzyme activity level at dawn but also contributed to a more efficient and successful defence against Botrytis cinerea infection.

OPDA/dn-OPDA actions: biosynthesis, metabolism, and signaling.

Plants cannot move, so they have evolved sophisticated strategies that integrate the external environmental cues and internal signaling networks for adaptation to dynamic circumstances. Cis-(+)-12-oxo-phytodienoic acid (OPDA) and 2,3-dinor-OPDA (dn-OPDA), the cyclopentenone-containing oxylipins, ubiquitously occur in the green lineage to orchestrate a series of growth and developmental processes as well as various stress and defense responses. OPDA/dn-OPDA are precursors of jasmonate (JA) biosynthesis in vascular plants. Dn-OPDA and its isomer also serve as bioactive JAs perceived by the coronatine insensitive 1/jasmonate ZIM-domain (COI1/JAZ) co-receptor complex in bryophytes and lycophytes. In addition, OPDA/dn-OPDA display signaling activities independent of (+)-7-iso-jasmonoyl-L-isoleucine (JA-Ile) and COI1 in both vascular and non-vascular plants. In this review, we discuss recent advances in the biosynthesis, metabolism, and signaling of OPDA/dn-OPDA, and provide an overview of the evolution of OPDA/dn-OPDA actions to obtain a deeper understanding of the pervasive role of OPDA/dn-OPDA in the plant life cycle.

BnVP1, a novel vacuolar H+ pyrophosphatase gene from Boehmeria nivea confers cadmium tolerance in transgenic Arabidopsis.

Plants have developed precise defense mechanisms against cadmium (Cd) stress, with vacuolar compartmentalization of Cd2+ being a crucial process in Cd detoxification. The transport of Cd into vacuoles by these cation / H+ antiporters is powered by the pH gradient created by proton pumps. In this study, the full-length cDNA of a vacuolar H+-pyrophosphatase (V-PPase) gene from Boehmeria nivea (ramie), BnVP1, was isolated using the rapid amplification of cDNA ends (RACE) method. The open reading frame (ORF) of BnVP1 is 2292 bp, encoding a 763 amino acid V-PPase protein with 15 predicted transmembrane domains. Sequence alignment and phylogenetic analysis revealed that BnVP1 belongs to the Type I V-PPase family. Quantitative RT-PCR assays demonstrated that BnVP1 expression was significantly higher in ramie roots than in shoots. Cd treatments markedly induced BnVP1 expression in both roots and leaves of ramie seedlings, with a more pronounced effect in roots. Additionally, BnVP1 expression was significantly upregulated by the plant hormone methyl jasmonate (MeJA). Heterologous expression of BnVP1 in transgenic Arabidopsis significantly enhanced V-PPase activity in the roots. The growth performance, root elongation, and total chlorophyll content of transgenic plants with high tonoplast H+-PPase (V-PPase) activity were superior to those of wild-type plants. Overexpression of BnVP1 reduced membrane lipid peroxidation and ion leakage, and significantly increased Cd accumulation in the roots of transgenic Arabidopsis seedlings. This study provides new genetic resources for the phytoremediation of Cd-contaminated farmland.

Early asymptomatic prediction of potato soft rot disease using phytohormone-induced volatile biomarkers.

Potatoes (Solanum tuberosum L.) are one of the world's major staple crops. In stored potatoes, Pectobacterium carotovorum subsp carotovorum causes soft rot. As a result of the rapid spread of the disease during post-harvest storage, potato production suffers huge losses. By detecting disease early and controlling it promptly, losses can be minimized. The profile of volatiles of plants can be altered by phytopathogens. Identifying unique volatile organic compounds (VOCs) as biomarkers for early disease detection has attracted considerable research attention. This study compared the VOC profiles of healthy and soft rot inoculated potatoes (cv. "Kufri Pukhraj") over a time course using gas chromatography-mass spectrometry (GC-MS). It was found that there was a differential emission of 27 VOCs between healthy non-inoculated potatoes and soft rot inoculated potatoes. Among 27 VOCs, only five (1-octen-3-ol, 2-methylisoborneol, 3-octanone, 1,4-dimethyladamantane, and 2-methyl-2-bornene) were found exclusively in soft rot inoculated potatoes, suggesting them potential biomarker for non-destructive prediction of soft rot disease in potatoes. Reactive oxygen species (H2O2) and phytohormone methyl-jasmonate (MeJa) levels increased transiently on infection with soft rot. The analysis of the primary metabolism of soft rot infected tubers at three different stages suggests metabolic reprogramming that occurs at the early stage of infection, possibly leading to biomarker volatile emission. Based on these results, it appears that the initial potato-soft rot bacteria interaction initiates metabolic reprogramming mainly through H2O2 and the MeJa signalling pathway. In asymptomatic potatoes, these biomarkers may be promising candidates for non-destructive detection of soft rot at an early stage. These biomarkers can be used to develop an e-nose sensor to predict soft rot in the future.

Functional analysis of two caffeoyl-coenzyme 3 a-o-methyltransferase involved in pear lignin metabolism.

Caffeoyl-coenzyme 3 A-O-methyltransferase (CCoAOMT) plays a crucial role in the lignin synthesis in many higher plants. In this study, nine PbCCoAOMT genes in total were identified from pear, and classified into six categories. We treated pear fruits with hormones abscisic acid (ABA) and methyl jasmonate (MeJA) and salicylic acid (SA) and observed differential expression levels of these genes. Through qRT-PCR, we also preliminarily identified candidate PbCCoAOMT gene, potentially involved in lignin synthesis in pear fruits. Additionally, the overexpression of PbCCoAOMT1/2 in Arabidopsis and pear fruits increased in lignin content. Enzymatic assays showed that recombinant PbCCoAOMT1/2 proteins have similar enzymatic activity in vitro. The Y1H (Yeast one-hybrid) and dual luciferase (dual-LUC) experiments demonstrated that PbMYB25 can bind to the AC elements in the promoter region of the PbCCoAOMT1 gene. Our findings suggested that the PbCCoAOMT1 and PbCCoAOMT2 genes may contribute to the synthesis of lignin and provide insights into the mechanism of lignin biosynthesis and stone cell development in pear fruits.

Improved pokeweed genome assembly and early gene expression changes in response to jasmonic acid.

BACKGROUND: Jasmonic acid (JA) is a phytohormone involved in regulating responses to biotic and abiotic stress. Although the JA pathway is well characterized in model plants such as Arabidopsis thaliana, less is known about many non-model plants. Phytolacca americana (pokeweed) is native to eastern North Americana and is resilient to environmental stress. The goal of this study was to produce a publicly available pokeweed genome assembly and annotations and use this resource to determine how early response to JA changes gene expression, with particular focus on genes involved in defense. RESULTS: We assembled the pokeweed genome de novo from approximately 30 Gb of PacBio Hifi long reads and achieved an NG50 of ~ 13.2 Mb and a minimum 93.9% complete BUSCO score for gene annotations. With this reference, we investigated the early changes in pokeweed gene expression following JA treatment. Approximately 5,100 genes were differentially expressed during the 0-6 h time course with almost equal number of genes with increased and decreased transcript levels. Cluster and gene ontology analyses indicated the downregulation of genes associated with photosynthesis and upregulation of genes involved in hormone signaling and defense. We identified orthologues of key transcription factors and constructed the first JA gene response network integrated with our transcriptomic data from orthologues of Arabidopsis genes. We discovered that pokeweed did not use leaf senescence as a means of reallocating resources during stress; rather, most secondary metabolite synthesis genes were constitutively expressed, suggesting that pokeweed directs its resources for survival over the long term. In addition, pokeweed synthesizes several RNA N-glycosylases hypothesized to function in defense, each with unique expression profiles in response to JA. CONCLUSIONS: Our investigation of the early response of pokeweed to JA illustrates patterns of gene expression involved in defence and stress tolerance. Pokeweed provides insight into the defense mechanisms of plants beyond those observed in research models and crops, and further study may yield novel approaches to improving the resilience of plants to environmental changes. Our assembled pokeweed genome is the first within the taxonomic family Phytolaccaceae to be publicly available for continued research.

QTL-seq identifies genomic region associated with the crown root development under Jasmonic acid response.

Rice root system plays a crucial role in plant adaptation under adverse conditions, particularly drought stress. However, the regulatory gene networks that govern rice root development during stress exposure remain largely unexplored. In this study, we applied a QTL sequencing method to identify QTL/gene controlling the crown root development under Jasmonic acid simulation using the Bulk-segregant analysis. Two rice cultivars with contrasting phenotypes from the Vietnamese traditional rice collection were used as parent pairs for crossing. The single-seed descent method was employed to generate an F2 population of progenies. This F2/3 population was further segregated based on root count under JA stress. Pooled DNA from the two extreme groups in this population was sequenced, and SNP indexes across all loci in these pools were calculated. We detected a significant genomic region on chromosome 10, spanned from 20.39-20.50 Mb, where two rice RLKs were located, OsPUB54 and OsPUB58. Receptor-like kinases (RLKs) are pivotal in regulating various aspects of root development in plants, and the U-box E3 ubiquitination ligase class was generally known for its degradation of some protein complexes. Notably, OsPUB54 was strongly induced by JA treatment, suggesting its involvement in the degradation of the Aux/IAA protein complex, thereby influencing crown root initiation. Besides, the Eukaryotic translation initiation of factor 3 subunit L (eIF3l) and the Mitogen-activated protein kinase kinase kinase 37 (MAPKKK 37) proteins identified from SNPs with high score index which suggests their significant roles in the translation initiation process and cellular signaling pathways, respectively. This information suggests several clues of how these candidates are involved in modifying the rice root system under stress conditions.

Sweet Immunity in Action: Unlocking Stem Reserves to Improve Yield and Quality. A Potential Key Role for Jasmonic Acid.

Common agronomic practices such as stem topping, side branch removal, and girdling can induce wound priming, mediated by jasmonic acid (JA). Low light conditions during greenhouse tomato production make the leaves more sensitive to the application of exogenous sugar, which is perceived as a "danger" in accordance with the concept of "Sweet Immunity". Consequently, source-sink balances are altered, leading to the remobilization of stem starch reserves and enabling the redirection of more carbon toward developing fruits, thereby increasing tomato yield and fruit quality. Similarities are drawn with the mobilization of fructans following defoliation of fodder grasses (wounding) and the remobilization of fructan and starch reserves under terminal drought and heat stress in wheat and rice (microwounding, cellular leakage). A central role for JA signaling is evident in all of these processes, closely intertwining with sugar signaling pathways. Therefore, JA signaling, associated with wounding and sugar priming events, offers numerous opportunities to alter source-sink balances across a broader spectrum of agricultural and horticultural crops, for instance, through the exogenous application of JA and fructans or a combination. This may entail reconfiguring and reversing phloem connections, potentially leading to an enhanced yield and product quality. Such processes may also disengage the growth-defense trade-off in plants.

Analysis of homodimer formation in 12-oxophytodienoate reductase 3 in solutio and crystallo challenges the physiological role of the dimer.

12-oxophytodienoate reductase 3 (OPR3) is a key enzyme in the biosynthesis of jasmonoyl-L-isoleucine, the receptor-active form of jasmonic acid and crucial signaling molecule in plant defense. OPR3 was initially crystallized as a self-inhibitory dimer, implying that homodimerization regulates enzymatic activity in response to biotic and abiotic stresses. Since a sulfate ion is bound to Y364, mimicking a phosphorylated tyrosine, it was suggested that dimer formation might be controlled by reversible phosphorylation of Y364 in vivo. To investigate OPR3 homodimerization and its potential physiological role in more detail, we performed analytical gel filtration and dynamic light scattering on wild-type OPR3 and three variants (R283D, R283E, and Y364P). The experiments revealed a rapid and highly sensitive monomer-dimer equilibrium for all OPR3 constructs. We crystallized all constructs with and without sulfate to examine its effect on the dimerization process and whether reversible phosphorylation of Y364 triggers homodimerization in vivo. All OPR3 constructs crystallized in their monomeric and dimeric forms independent of the presence of sulfate. Even variant Y364P, lacking the putative phosphorylation site, was crystallized as a self-inhibitory homodimer, indicating that Y364 is not required for dimerization. Generally, the homodimer is relatively weak, and our results raise doubts about its physiological role in regulating jasmonate biosynthesis.

Characterization of the ZCTs, a subgroup of Cys2-His2 zinc finger transcription factors regulating alkaloid biosynthesis in Catharanthus roseus.

KEY MESSAGE: The C. roseus ZCTs are jasmonate-responsive, can be induced by CrMYC2a, and can act as significant regulators of the terpenoid indole alkaloid pathway when highly expressed. Catharanthus roseus is the sole known producer of the anti-cancer terpenoid indole alkaloids (TIAs), vinblastine and vincristine. While the enzymatic steps of the pathway have been elucidated, an understanding of its regulation is still emerging. The present study characterizes an important subgroup of Cys2-His2 zinc finger transcription factors known as Zinc finger Catharanthus Transcription factors (ZCTs). We identified three new ZCT members (named ZCT4, ZCT5, and ZCT6) that clustered with the putative repressors of the TIA pathway, ZCT1, ZCT2, and ZCT3. We characterized the role of these six ZCTs as potential redundant regulators of the TIA pathway, and their tissue-specific and jasmonate-responsive expression. These ZCTs share high sequence conservation in their two Cys2-His2 zinc finger domains but differ in the spacer length and sequence between these zinc fingers. The transient overexpression of ZCTs in seedlings significantly repressed the promoters of the terpenoid (pLAMT) and condensation branch (pSTR1) of the TIA pathway, consistent with that previously reported for ZCT1, ZCT2, and ZCT3. In addition, ZCTs significantly repressed and indirectly activated several promoters of the vindoline pathway (not previously studied). The ZCTs differed in their tissue-specific expression but similarly increased with jasmonate in a dosage-dependent manner (except for ZCT5). We showed significant activation of the pZCT1 and pZCT3 promoters by the de-repressed CrMYC2a, suggesting that the jasmonate-responsive expression of the ZCTs can be mediated by CrMYC2a. In summary, the C. roseus ZCTs are jasmonate-responsive, can be induced by CrMYC2a, and can act as significant regulators of the TIA pathway when highly expressed.

The development of yellow lupin anthers depends on the relationship between jasmonic acid and indole-3-acetic acid.

The main purpose of this study was to demonstrate that the course of anther development, including post-meiotic maturation, dehiscence and senescence, is ensured by the interdependencies between jasmonic acid (JA) and indole-3-acetic acid (IAA) in yellow lupin (Lupinus luteus L.). The concentration of JA peaked during anther dehiscence when IAA level was low, whereas the inverse relationship was specific to anther senescence. Cellular and tissue localization of JA and IAA, in conjunction with broad expression profile for genes involved in biosynthesis, signalling, response, and homeostasis under different conditions, allowed to complete and define the role of studied phytohormones during late anther development, as well as predict events triggered by them. The development/degeneration of septum and anther wall cells, dehydration of epidermis, and rupture of stomium may involve JA signalling, while the formation of secondary thickening in endothecial cell walls is rather JA independent. The IAA is involved in programmed cell death (PCD)-associated processes during anther senescence but does not exclude its participation in the anther dehiscence processes, mainly related to cell disintegration and degeneration. A detailed understanding of these multistage processes, especially at the level of phytohormonal interplay, can contribute to the effective control of male fertility, potentially revolutionizing the breeding of L. luteus.