Neofunctionalization of an OMT cluster dominates polymethoxyflavone biosynthesis associated with the domestication of citrus.

Polymethoxyflavones (PMFs) are a class of abundant specialized metabolites with remarkable anticancer properties in citrus. Multiple methoxy groups in PMFs are derived from methylation modification catalyzed by a series of hydroxylases and O-methyltransferases (OMTs). However, the specific OMTs that catalyze the systematic O-methylation of hydroxyflavones remain largely unknown. Here, we report that PMFs are highly accumulated in wild mandarins and mandarin-derived accessions, while undetectable in early-diverging citrus species and related species. Our results demonstrated that three homologous genes, CreOMT3, CreOMT4, and CreOMT5, are crucial for PMF biosynthesis in citrus, and their encoded methyltransferases exhibit multisite O-methylation activities for hydroxyflavones, producing seven PMFs in vitro and in vivo. Comparative genomic and syntenic analyses indicated that the tandem CreOMT3, CreOMT4, and CreOMT5 may be duplicated from CreOMT6 and contributes to the genetic basis of PMF biosynthesis in the mandarin group through neofunctionalization. We also demonstrated that N17 in CreOMT4 is an essential amino acid residue for C3-, C5-, C6-, and C3'-O-methylation activity and provided a rationale for the functional deficiency of OMT6 to produce PMFs in early-diverging citrus and some domesticated citrus species. A 1,041-bp deletion in the CreOMT4 promoter, which is found in most modern cultivated mandarins, has reduced the PMF content relative to that in wild and early-admixture mandarins. This study provides a framework for reconstructing PMF biosynthetic pathways, which may facilitate the breeding of citrus fruits with enhanced health benefits.

Multi-omics analyses reveal the importance of chromoplast plastoglobules in carotenoid accumulation in citrus fruit.

Chromoplasts act as a metabolic sink for carotenoids, in which plastoglobules serve as versatile lipoprotein particles. PGs in chloroplasts have been characterized. However, the features of PGs from non-photosynthetic plastids are poorly understood. We found that the development of chromoplast plastoglobules (CPGs) in globular and crystalloid chromoplasts of citrus is associated with alterations in carotenoid storage. Using Nycodenz density gradient ultracentrifugation, an efficient protocol for isolating highly purified CPGs from sweet orange (Citrus sinensis) pulp was established. Forty-four proteins were defined as likely comprise the core proteome of CPGs using comparative proteomics analysis. Lipidome analysis of different chromoplast microcompartments revealed that the nonpolar microenvironment within CPGs was modified by 35 triacylglycerides, two sitosterol esters, and one stigmasterol ester. Manipulation of the CPG-localized gene CsELT1 (esterase/lipase/thioesterase) in citrus calli resulted in increased lipids and carotenoids, which is further evidence that the nonpolar microenvironment of CPGs contributes to carotenoid accumulation and storage in the chromoplasts. This multi-feature analysis of CPGs sheds new light on the role of chromoplasts in carotenoid metabolism, paving the way for manipulating carotenoid content in citrus fruit and other crops.

Adventitious embryonic causal gene FhRWP regulates multiple developmental phenotypes in citrus reproduction.

Citrus is a model plant for studying adventitious embryos, a form of asexual reproduction controlled by a single dominant gene, RWP. This gene has been identified as the causal gene for nucellar embryogenesis, but its function has not yet been fully understood. In this study, we used the fast-growing Fortunella hindsii as a system to explore chromatin accessibility during the nucellar embryony initiation, emphasizing elevated chromatin accessibility in polyembryonic (PO) genotypes compared to monoembryonic ones (MO). Notably, a higher level of accessible chromatin was observed in one allele of the promoter region of FhRWP, consistent with increased expression of the allele carrying the causal structural variant. By independently performing RNAi and gene editing experiments on PO genotypes, we found the downregulation of FhRWP expression could reduce the number of nucellar embryos, while its knockout resulted in abnormal axillary bud development. In overexpression experiments, FhRWP was identified as having the unique capability of inducing the embryogenic callus formation in MO stem segments, possibly through the regulation of the WUS-CLV signaling network and the ABA and cytokinin pathway, marking the inaugural demonstration of FhRWP's potential to reignite somatic cells' embryogenic fate. This study reveals the pleiotropic function of RWP in citrus and constructs a regulatory network during adventitious embryo formation, providing a new tool for bioengineering applications in plant regeneration.

The CsAP2-09-CsWRKY25-CsRBOH2 cascade confers resistance against citrus bacterial canker by regulating ROS homeostasis.

Citrus bacterial canker (CBC) is a serious bacterial disease caused by Xanthomonas citri subsp. citri (Xcc) that adversely impacts the global citrus industry. In a previous study, we demonstrated that overexpression of an Xcc-inducible apetala 2/ethylene response factor encoded by Citrus sinensis, CsAP2-09, enhances CBC resistance. The mechanism responsible for this effect, however, is not known. In the present study, we showed that CsAP2-09 targeted the promoter of the Xcc-inducible WRKY transcription factor coding gene CsWRKY25 directly, activating its transcription. CsWRKY25 was found to localize to the nucleus and to activate transcriptional activity. Plants overexpressing CsWRKY25 were more resistant to CBC and showed higher expression of the respiratory burst oxidase homolog (RBOH) CsRBOH2, in addition to exhibiting increased RBOH activity. Transient overexpression assays in citrus confirmed that CsWRKY25 and CsRBOH2 participated in the generation of reactive oxygen species (ROS) bursts, which were able to restore the ROS degradation caused by CsAP2-09 knockdown. Moreover, CsWRKY25 was found to bind directly to W-box elements within the CsRBOH2 promoter. Notably, CsRBOH2 knockdown had been reported previously to reduce the CBC resistance, while demonstrated in this study, CsRBOH2 transient overexpression can enhance the CBC resistance. Overall, our results outline a pathway through which CsAP2-09-CsWRKY25 transcriptionally reprograms CsRBOH2-mediated ROS homeostasis in a manner conducive to CBC resistance. These data offer new insight into the mechanisms and regulatory pathways through which CsAP2-09 regulates CBC resistance, highlighting its potential utility as a target for the breeding of CBC-resistant citrus varieties.

Molecular basis of one-step methyl anthranilate biosynthesis in grapes, sweet orange, and maize.

Plants synthesize an array of volatile compounds, many of which serve ecological roles in attracting pollinators, deterring herbivores, and communicating with their surroundings. Methyl anthranilate (MeAA) is an anti-herbivory defensive volatile responsible for grape aroma that is emitted by several agriculturally relevant plants, including citrus, grapes, and maize. Unlike maize, which uses a one-step anthranilate methyltransferase (AAMT), grapes have been thought to use a two-step pathway for MeAA biosynthesis. By mining available transcriptomics data, we identified two AAMTs in Vitis vinifera (wine grape), as well as one ortholog in "Concord" grape. Many angiosperms methylate the plant hormone salicylic acid (SA) to produce methyl salicylate, which acts as a plant-to-plant communication molecule. Because the Citrus sinensis (sweet orange) SA methyltransferase can methylate both anthranilate (AA) and SA, we used this enzyme to examine the molecular basis of AA activity by introducing rational mutations, which identified several active site residues that increase activity with AA. Reversing this approach, we introduced mutations that imparted activity with SA in the maize AAMT, which uncovered different active site residues from those in the citrus enzyme. Sequence and phylogenetic analysis revealed that one of the Vitis AAMTs shares an ancestor with jasmonic acid methyltransferases, similar to the AAMT from strawberry (Frageria sp.). Collectively, these data demonstrate the molecular mechanisms underpinning AA activity across methyltransferases and identify one-step enzymes by which grapes synthesize MeAA.

Transcriptome and metabolome atlas reveals contributions of sphingosine and chlorogenic acid to cold tolerance in Citrus.

Citrus is one of the most important fruit crop genera in the world, but many Citrus species are vulnerable to cold stress. Ichang papeda (Citrus ichangensis), a cold-hardy citrus species, holds great potential for identifying valuable metabolites that are critical for cold tolerance in Citrus. However, the metabolic changes and underlying mechanisms that regulate Ichang papeda cold tolerance remain largely unknown. In this study, we compared the metabolomes and transcriptomes of Ichang papeda and HB pummelo (Citrus grandis 'Hirado Buntan', a cold-sensitive species) to explore the critical metabolites and genes responsible for cold tolerance. Metabolomic analyses led to the identification of common and genotype-specific metabolites, consistent with transcriptomic alterations. Compared to HB pummelo under cold stress, Ichang papeda accumulated more sugars, flavonoids, and unsaturated fatty acids, which are well-characterized metabolites involved in stress responses. Interestingly, sphingosine and chlorogenic acid substantially accumulated only in Ichang papeda. Knockdown of CiSPT (C. ichangensis serine palmitoyltransferase) and CiHCT2 (C. ichangensis hydroxycinnamoyl-CoA: shikimate hydroxycinnamoyltransferase2), two genes involved in sphingosine and chlorogenic acid biosynthesis, dramatically decreased endogenous sphingosine and chlorogenic acid levels, respectively. This reduction in sphingosine and chlorogenic acid notably compromised the cold tolerance of Ichang papeda, whereas exogenous application of these metabolites increased plant cold tolerance. Taken together, our findings indicate that greater accumulation of a spectrum of metabolites, particularly sphingosine and chlorogenic acid, promotes cold tolerance in cold-tolerant citrus species. These findings broaden our understanding of plant metabolic alterations in response to cold stress and provide valuable targets that can be manipulated to improve Citrus cold tolerance.

Pan-mitogenomics reveals the genetic basis of cytonuclear conflicts in citrus hybridization, domestication, and diversification.

Although interactions between the cytoplasmic and nuclear genomes occurred during diversification of many plants, the evolutionary conflicts due to cytonuclear interactions are poorly understood in crop breeding. Here, we constructed a pan-mitogenome and identified chimeric open reading frames (ORFs) generated by extensive structural variations (SVs). Meanwhile, short reads from 184 accessions of citrus species were combined to construct three variation maps for the nuclear, mitochondrial, and chloroplast genomes. The population genomic data showed discordant topologies between the cytoplasmic and nuclear genomes because of differences in mutation rates and levels of heteroplasmy from paternal leakage. An analysis of species-specific SVs indicated that mitochondrial heteroplasmy was common and that chloroplast heteroplasmy was undetectable. Interestingly, we found a prominent divergence in the mitogenomes and the highest genetic load in the, which may provide the basis for cytoplasmic male sterility (CMS) and thus influence the reshuffling of the cytoplasmic and nuclear genomes during hybridization. Using cytoplasmic replacement experiments, we identified a type of species-specific CMS in mandarin related to two chimeric mitochondrial genes. Our analyses indicate that cytoplasmic genomes from mandarin have rarely been maintained in hybrids and that paternal leakage produced very low levels of mitochondrial heteroplasmy in mandarin. A genome-wide association study (GWAS) provided evidence for three nuclear genes that encode pentatricopeptide repeat (PPR) proteins contributing to the cytonuclear interactions in the Citrus genus. Our study demonstrates the occurrence of evolutionary conflicts between cytoplasmic and nuclear genomes in citrus and has important implications for genetics and breeding.

Longitudinal Transcriptomic, Proteomic, and Metabolomic Response of Citrus sinensis to Diaphorina citri Inoculation of Candidatus Liberibacter asiaticus.

Huanglongbing (HLB) is a fatal citrus disease that is currently threatening citrus varieties worldwide. One putative causative agent, Candidatus Liberibacter asiaticus (CLas), is vectored by Diaphorina citri, known as the Asian citrus psyllid (ACP). Understanding the details of CLas infection in HLB disease has been hindered by its Candidatus nature and the inability to confidently detect it in diseased trees during the asymptomatic stage. To identify early changes in citrus metabolism in response to inoculation of CLas using its natural psyllid vector, leaves from Madam Vinous sweet orange (Citrus sinensis (L.) Osbeck) trees were exposed to CLas-positive ACP or CLas-negative ACP and longitudinally analyzed using transcriptomics (RNA sequencing), proteomics (liquid chromatography-tandem mass spectrometry; data available in Dryad: 10.25338/B83H1Z), and metabolomics (proton nuclear magnetic resonance). At 4 weeks postexposure (wpe) to psyllids, the initial HLB plant response was primarily to the ACP and, to a lesser extent, the presence or absence of CLas. Additionally, analysis of 4, 8, 12, and 16 wpe identified 17 genes and one protein as consistently differentially expressed between leaves exposed to CLas-positive ACP versus CLas-negative ACP. This study informs identification of early detection molecular targets and contributes to a broader understanding of vector-transmitted plant pathogen interactions.

Laser capture microdissection-based spatiotemporal transcriptomes uncover regulatory networks during seed abortion in seedless Ponkan (Citrus reticulata).

Seed abortion is an important process in the formation of seedless characteristics in citrus fruits. However, the molecular regulatory mechanism underlying citrus seed abortion is poorly understood. Laser capture microdissection-based RNA-seq combined with Pacbio-seq was used to profile seed development in the Ponkan cultivars 'Huagan No. 4' (seedless Ponkan) (Citrus reticulata) and 'E'gan No. 1' (seeded Ponkan) (C. reticulata) in two types of seed tissue across three developmental stages. Through comparative transcriptome and dynamic phytohormone analyses, plant hormone signal, cell division and nutrient metabolism-related processes were revealed to play critical roles in the seed abortion of 'Huagan No. 4'. Moreover, several genes may play indispensable roles in seed abortion of 'Huagan No. 4', such as CrWRKY74, CrWRKY48 and CrMYB3R4. Overexpression of CrWRKY74 in Arabidopsis resulted in severe seed abortion. By analyzing the downstream regulatory network, we further determined that CrWRKY74 participated in seed abortion regulation by inducing abnormal programmed cell death. Of particular importance is that a preliminary model was proposed to depict the regulatory networks underlying seed abortion in citrus. The results of this study provide novel insights into the molecular mechanism across citrus seed development, and reveal the master role of CrWRKY74 in seed abortion of 'Huagan No. 4'.

A Calcium-Dependent Protein Kinase Regulates the Defense Response in Citrus sinensis.

Citrus Huanglongbing (HLB), which is caused by 'Candidatus Liberibacter asiaticus' (CLas), is one of the most destructive citrus diseases worldwide, and defense-related Citrus sinensis gene resources remain largely unexplored. Calcium signaling plays an important role in diverse biological processes. In plants, a few calcium-dependent protein kinases (CDPKs/CPKs) have been shown to contribute to defense against pathogenic microbes. The genome of C. sinensis encodes dozens of CPKs. In this study, the role of C. sinensis calcium-dependent protein kinases (CsCPKs) in C. sinensis defense was investigated. Silencing of CsCPK6 compromised the induction of defense-related genes in C. sinensis. Expression of a constitutively active form of CsCPK6 (CsCPK6CA) triggered the activation of defense-related genes in C. sinensis. Complementation of CsCPK6 rescued the defense-related gene induction in an Arabidopsis thaliana cpk4/11 mutant, indicating that CsCPK6 carries CPK activity and is capable of functioning as a CPK in Arabidopsis. Moreover, an effector derived from CLas inhibits defense induced by the expression of CsCPK6CA and autophosphorylation of CsCPK6, which suggests the involvement of CsCPK6 and calcium signaling in defense. These results support a positive role for CsCPK6 in C. sinensis defense against CLas, and the autoinhibitory regulation of CsCPK6 provides a potential genome-editing target for improving C. sinensis defense. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

OrangeExpDB: an integrative gene expression database for Citrus spp.

BACKGROUND: Citrus is a major fruit crop, and RNA-sequencing (RNA-seq) data can be utilized to investigate its gene functions, heredity, evolution, development, and the detection of genes linked to essential traits or resistance to pathogens. However, it is challenging to use the public RNA-seq datasets for researchers without bioinformatics training, and expertise. RESULTS: OrangeExpDB is a web-based database that integrates transcriptome data of various Citrus spp., including C. limon (L.) Burm., C. maxima (Burm.) Merr., C. reticulata Blanco, C. sinensis (L.) Osbeck, and Poncirus trifoliata (L.) Raf., downloaded from the NCBI SRA database. It features a blast tool for browsing and searching, enabling quick download of expression matrices for different transcriptome samples. Expression of genes of interest can be easily generated by searching gene IDs or sequence similarity. Expression data in text format can be downloaded and presented as a heatmap, with additional sample information provided at the bottom of the webpage. CONCLUSIONS: Researchers can utilize OrangeExpDB to facilitate functional genomic analysis and identify key candidate genes, leveraging publicly available citrus RNA-seq datasets. OrangeExpDB can be accessed at http://www.orangeexpdb.com/ .

Genome-wide identification of the GRF family in sweet orange (Citrus sinensis) and functional analysis of the CsGRF04 in response to multiple abiotic stresses.

BACKGROUND: Citrus is one of the most valuable fruits worldwide and an economic pillar industry in southern China. Nevertheless, it frequently suffers from undesirable environmental stresses during the growth cycle, which severely restricts the growth, development and yield of citrus. In plants, the growth-regulating factor (GRF) family of transcription factors (TF) is extensively distributed and plays an vital part in plant growth and development, hormone response, as well as stress adaptation. However, the systematic identification and functional analysis of GRF TFs in citrus have not been reported. RESULTS: Here, a genome-wide identification of GRF TFs was performed in Citrus sinensis, 9 members of CsGRFs were systematically identified and discovered to be scattered throughout 5 chromosomes. Subsequently, physical and chemical properties, phylogenetic relationships, structural characteristics, gene duplication events, collinearity and cis-elements of promoter were elaborately analyzed. In particular, the expression patterns of the CsGRF genes in response to multiple phytohormone and abiotic stress treatments were investigated. Predicated on this result, CsGRF04, which exhibited the most differential expression pattern under multiple phytohormone and abiotic stress treatments was screened out. Virus-induced gene silencing (VIGS) technology was utilized to obtain gene silenced plants for CsGRF04 successfully. After the three stress treatments of high salinity, low temperature and drought, the CsGRF04-VIGS lines showed significantly reduced resistance to high salinity and low temperature stresses, but extremely increased resistance to drought stress. CONCLUSIONS: Taken together, our findings systematically analyzed the genomic characterization of GRF family in Citrus sinensis, and excavated a CsGRF04 with potential functions under multiple abiotic stresses. Our study lay a foundation for further study on the function of CsGRFs in abiotic stress and hormone signaling response.

Involvement of CgHSFB1 in the regulation of self-incompatibility in 'Shatian' pummelo.

As self-incompatibility is a major issue in pummelo breeding and production, its mechanism in citrus was analyzed to improve breeding efficiency and reduce production costs. Rutaceae belongs to S-RNase type of gametophytic self-incompatibility. While the function of S-RNase/SLF and the mechanism of self-incompatibility have been studied extensively, the transcriptional regulation of S-RNase has been less studied. We performed transcriptome sequencing with the styles of 'Shatian' pummelo on the day of anthesis and 1-5 days before anthesis, and found that the transcript level of S-RNase gradually decreased with flower development. By analyzing differentially expressed genes and correlation with the expression trend of S-RNase, we identified a candidate gene, CgHSFB1, and utilized biochemical experiments such as yeast one-hybrid assay, electrophoretic mobility shift assay and dual-luciferase assay, as well as transient transformation of citrus calli and Citrus microcarpa and demonstrated that CgHSFB1 could directly bind to the S1-RNase promoter and repress the expression of S1-RNase, which is involved in the pummelo self-incompatibility response. In contrast, CgHSFB1 did not bind to the promoter of S2-RNase, and there was specificity in the regulation of S-RNase.

Inhibition of Glial Activation and Subsequent Reduction in White Matter Damage through Supplementation with a Combined Extract of Wheat Bran, Citrus Peel, and Jujube in a Rat Model of Vascular Dementia.

Vascular dementia (VaD) is the second most common type of dementia after Alzheimer's disease. In our previous studies, we showed that wheat bran extract (WBE) reduced white matter damage in a rat VaD model and improved memory in a human clinical trial. However, starch gelatinization made the large-scale preparation of WBE difficult. To simplify the manufacturing process and increase efficacy, we attempted to find a decoction containing an optimum ratio of wheat bran, sliced citrus peel, and sliced jujube (WCJ). To find an optimal ratio, the cell survival of C6 (rat glioma) cultured under hypoxic conditions (1% O2) was measured, and apoptosis was assessed. To confirm the efficacies of the optimized WCJ for VaD, pupillary light reflex, white matter damage, and the activation of astrocytes and microglia were assessed in a rat model of bilateral common carotid artery occlusion (BCCAO) causing chronic hypoperfusion. Using a combination of both searching the literature and cell survival experiments, we chose 6:2:1 as the optimal ratio of wheat bran to sliced citrus peel to sliced jujube to prepare WCJ. We showed that phytic acid contained only in wheat bran can be used as an indicator component for the quality control of WCJ. We observed in vitro that the WCJ treatment improved cell survival by reducing apoptosis through an increase in the Bcl-2/Bax ratio. In the BCCAO experiments, the WCJ-supplemented diet prevented astrocytic and microglial activation, mitigated myelin damage in the corpus callosum and optic tract, and, consequently, improved pupillary light reflex at dosages over 100 mg/kg/day. The results suggest that the consumption of WCJ can prevent VaD by reducing white matter damage, and WCJ can be developed as a safe, herbal medicine to prevent VaD.

D-Limonene reduces depression-like behaviour and enhances learning and memory through an anti-neuroinflammatory mechanism in male rats subjected to chronic restraint stress.

D-limonene is a widely used flavouring additive in foods, beverages and fragrances due to its pleasant lemon-like odour. This study aimed to investigate the effects of D-limonene on the central nervous system when subjected to chronic restraint stress in rats for 21 days. Forty rats were randomly divided into five groups: i) control, ii) D-limonene, iii) restraint stress, iv) restraint stress+D-limonene and v) restraint stress+fluoxetine. Following the induction of restraint stress, the sucrose preference test, the open field test, the novel object recognition test and the forced swimming test were performed. The levels of BDNF, IL-1ß, IL-6 and caspase-1 were measured from hippocampal tissue using the ELISA method. Sucrose preference test results showed an increase in consumption rate in the stress+D-limonene and a decrease in the stress group. The stress+D-limonene group reversed the increased defensive behaviour observed in the open-field test compared to the stress group. In the novel object recognition test, the discrimination index of the stress+D-limonene group increased compared to the stress group. BDNF levels increased in the stress+limonene group compared to the stress group. In contrast, IL-1ß and caspase-1 levels increased in the stress group compared to the control and decreased in the stress+limonene group compared to the stress group. In this study, D-limonene has been found to have antidepressant-like properties, reducing anhedonic and defensive behaviours and the impairing effects of stress on learning and memory tests. It was observed that D-limonene showed these effects by alleviating neuroinflammation induced by chronic restraint stress in rats.

Characterization and biological prospects of various calcined temperature-V2O5 nanoparticles synthesized by Citrus hystrix fruit extract.

In this study, a simple green synthesis of vanadium pentoxide nanoparticles (VNPs) was prepared by the extract of Kaffir lime fruit (Citrus hystrix) as a green reducing and stabilizing agent, along with the investigation of calcination temperature was carried out at 450 and 550 °C. It was affirmed that, at higher temperature (550 °C), the VNPs possessed a high degree crystalline following the construction of (001) lattice diffraction within an increase in crystalline size from 47.12 to 53.51 nm, although the band gap of the materials at 450 °C was lower than that of the VNPs-550 (2.53 versus 2.66 eV, respectively). Besides, the materials were assessed for the potential bioactivities toward antibacterial, antifungal, DNA cleavage, anti-inflammatory, and hemolytic performances. As a result, the antibacterial activity, with minimal inhalation concentration (MIC) < 6.25 µg/mL for both strains, and fungicidal one of the materials depicted the dose-dependent effects. Once, both VNPs exhibited the noticeable efficacy of the DNA microbial damage, meanwhile, the outstanding anti-inflammatory agent was involved with the IC50 of 123.636 and 227.706 µg/mL, accounting for VNPs-450 and VNPs-550, respectively. Furthermore, this study also demonstrated the hemolytic potential of the VNPs materials. These consequences declare the prospects of the VNPs as the smart and alternative material from the green procedure in biomedicine.

Deciphering the drug delivery potential of Type1 lipid transfer protein from Citrus sinensis for enhancing the therapeutic efficacy of drugs.

Type1 Non-specific Lipid Transfer Protein (CsLTP1) from Citrus sinensis is a small cationic protein possessing a long tunnel-like hydrophobic cavity. CsLTP1 performing membrane trafficking of lipids is a promising candidate for developing a potent drug delivery system. The present work includes in-silico studies and the evaluation of drugs binding to CsLTP1 using biophysical techniques along with the investigation of CsLTP1's ability to enhance the efficacy of drugs employing cell-based bioassays. The in-silico investigations identified Panobinostat, Vorinostat, Cetylpyridinium Chloride, and Fulvestrant with higher affinities and stability of binding to the hydrophobic pocket of CsLTP1. SPR studies revealed strong binding affinities of anticancer drugs, Panobinostat (KD = 1.40 µM) and Vorinostat (KD = 2.17 µM) to CsLTP1 along with the binding and release kinetics. CD and fluorescent spectroscopy revealed drug-induced conformational changes in CsLTP1. CsLTP1-associated drug forms showed remarkably enhanced efficacy in MCF-7 cells, representing increased cell cytotoxicity, intracellular ROS, reduced mitochondrial membrane potential, and up-regulation of proapoptotic markers than the free drugs employing qRT-PCR and western blot analysis. The findings demonstrate that CsLTP1 binds strongly to hydrophobic drugs to facilitate their transport, hence improving their therapeutic efficacy revealed by the in-vitro investigations. This study establishes an excellent foundation for developing CsLTP1-based efficient drug delivery system.

Comparative mineral and biochemical characterization of Citrus reticulata fruits and leaves to citrus canker pathogens, Xanthomonas axonopodis.

Pakistan's economy greatly benefits from citrus production since these fruits are sold and consumed all over the world. Although citrus fruits are easy to cultivate, they are susceptible to diseases caused by bacteria, viruses, and fungi. These challenges, as well as difficulties in obtaining the proper nutrients, might negatively impact fruit yields and quality. Citrus canker is another complicated problem caused by the germ Xanthomonas axonopodis. This germ affects many types of citrus fruits all over the world. This study looked closely at how citrus canker affects the leaves and the quality of the fruit in places like Sargodha, Bhalwal, Kotmomin, and Silanwali, which are big areas for growing citrus in the Sargodha district. What we found was that plants without the disease had more chlorophyll in their leaves compared to the sick plants. Also, the healthy plants had better amounts of important minerals like calcium, magnesium, potassium, and phosphorus in their fruits. But the fruits with the disease had too much sodium, and the iron levels were a bit different. The fruits with the disease also didn't have as much of something that protects them called antioxidants, which made them more likely to get sick. This study helps us understand how citrus canker affects plants and fruit, so we can think of ways to deal with it.

Genome-wide identification, expression analysis of WRKY transcription factors in Citrus ichangensis and functional validation of CiWRKY31 in response to cold stress.

BACKGROUND: Ichang papeda (Citrus ichangensis), a wild perennial plant of the Rutaceae family, is a cold-hardy plant. WRKY transcription factors are crucial regulators of plant growth and development as well as abiotic stress responses. However, the WRKY genes in C. ichangensis (CiWRKY) and their expression patterns under cold stress have not been thoroughly investigated, hindering our understanding of their role in cold tolerance. RESULTS: In this study, a total of 52 CiWRKY genes identified in the genome of C. ichangensis were classified into three main groups and five subgroups based on phylogenetic analysis. Comprehensive analyses of motif features, conserved domains, and gene structures were performed. Segmental duplication plays a significant role in the CiWRKY gene family expansion. Cis-acting element analysis revealed the presence of various stress-responsive elements in the promoters of the majority of CiWRKYs. Gene ontology (GO) analysis and protein-protein interaction predictions indicate that the CiWRKYs exhibit crucial roles in regulation of both development and stress response. Expression profiling analysis demonstrates that 14 CiWRKYs were substantially induced under cold stress. Virus-induced gene silencing (VIGS) assay confirmed that CiWRKY31, one of the cold-induced WRKYs, functions positively in regulation of cold tolerance. CONCLUSION: Sequence and protein properties of CiWRKYs were systematically analyzed. Among the 52 CiWRKY genes 14 members exhibited cold-responsive expression patterns, and CiWRKY31 was verified to be a positive regulator of cold tolerance. These findings pave way for future investigations to understand the molecular functions of CiWRKYs in cold tolerance and contribute to unravelling WRKYs that may be used for engineering cold tolerance in citrus.

QTL analysis on a lemon population provides novel insights on the genetic regulation of the tolerance to the two-spotted spider mite attack.

BACKGROUND: Among the Citrus species, lemon (Citrus limon Burm f.) is one of the most affected by the two-spotted spider mite (Tetranychus urticae Koch). Moreover, chemical control is hampered by the mite's ability to develop genetic resistance against acaricides. In this context, the identification of the genetic basis of the host resistance could represent a sustainable strategy for spider mite control. In the present study, a marker-trait association analysis was performed on a lemon population employing an association mapping approach. An inter-specific full-sib population composed of 109 accessions was phenotyped through a detached-leaf assays performed in modified Huffaker cells. Those individuals, complemented with two inter-specific segregating populations, were genotyped using a target-sequencing approach called SPET (Single Primer Enrichment Technology), the resulting SNPs were employed for the generation of an integrated genetic map. RESULTS: The percentage of damaged area in the full-sib population showed a quantitative distribution with values ranging from 0.36 to 9.67%. A total of 47,298 SNPs were selected for an association mapping study and a significant marker linked with resistance to spider mite was detected on linkage group 5. In silico gene annotation of the QTL interval enabled the detection of 13 genes involved in immune response to biotic and abiotic stress. Gene expression analysis showed an over expression of the gene encoding for the ethylene-responsive transcription factor ERF098-like, already characterized in Arabidopsis and in rice for its involvement in defense response. CONCLUSION: The identification of a molecular marker linked to the resistance to spider mite attack can pave the way for the development of marker-assisted breeding plan for the development of novel selection coupling favorable agronomical traits (e.g. fruit quality, yield) with a higher resistance toward the mite.