Screening for antibacterial and cytotoxic activities of Sri Lankan marine sponges through microfractionation: Isolation of bromopyrrole alkaloids from Stylissa massa.

Sri Lanka is a biodiversity hotspot and one of the richest geographical locations of marine sponges in the Indian ocean. However, the most extensive taxonomical study on Sri Lankan sponge biodiversity dates back ~100 years and only a limited number of studies have been conducted on sponge natural products. In the current study, 35 marine sponge specimens (collected from 16 sponge habitats around Sri Lanka) were identified, microfractionated and evaluated for antibacterial and anticancer assays. In total, 30 species were characterized, of which 19 species gave extracts with antibacterial and/or cytotoxic activities. Microfractionated organic extract of Aciculites orientalis gave the most potent antibacterial activity against Staphylococcus aureus and strongest lymphoma cell toxicity was exhibited by the organic extract of Acanthella sp. Guided by the molecular ion peaks in the bioactive fractions, large-scale extraction of Stylissa massa led to the isolation of three bromopyrrole alkaloids, sceptrin, hymenin and manzacidin A/C. Of these, sceptrin exhibited broad spectrum antibacterial activity against both Escherichia coli and S. aureus (MIC of 62.5 µM against both species). Based on natural product literature, seven promising species were identified as understudied. Their further exploration may lead to the discovery of structurally novel compounds.

Antibacterial eremophilane sesquiterpenoids from Xylaria feejeensis, an endophytic fungi of the medicinal plant Geophila repens.

Geophila repens (L.) I.M. Johnst (Rubiaceae) is a traditional medicinal plant used in Sri Lanka for the treatment of bacterial infections. Due to its rich endophytic fungi content, it was postulated that endophytically-produced specialized metabolites may be responsible for its purported antibacterial effects. To test this hypothesis, eight pure endophytic fungal cultures were isolated from G. repens then extracted and screened for antibacterial activity in a disc diffusion assay against Staphylococcus aureus, Bacillus cereus, Escherichia coli and Pseudomonas aeruginosa. Large scale culturing, extraction, and purification of the most active fungal extract, obtained from Xylaria feejeensis, led to the isolation of 6',7'-didehydrointegric acid (1), 13-carboxyintegric acid (2), and four known compounds including integric acid (3). Compound 3 was isolated as the key antibacterial component (MIC = 16 µg/mL against Bacillus subtilis, 64 µg/mL against Methicillin-Resistant S. aureus). Compound 3 and its analogues were devoid of hemolytic activity up to the highest tested concentration of 45 µg/mL. This study demonstrates that specialized metabolites produced by endophytic fungi may contribute to the biological activity of some medicinal plants. Endophytic fungi should be evaluated as a potential source of antibiotics, especially from unexplored medicinal plants traditionally used for the treatment of bacterial infections.

Screening for Cyclotides in Sri Lankan Medicinal Plants: Discovery, Characterization, and Bioactivity Screening of Cyclotides from Geophila repens.

Cyclotides are an intriguing class of structurally stable circular miniproteins of plant origin with numerous potential pharmaceutical and agricultural applications. To investigate the occurrence of cyclotides in Sri Lankan flora, 50 medicinal plants were screened, leading to the identification of a suite of new cyclotides from Geophila repens of the family Rubiaceae. Cycloviolacin O2-like (cyO2-like) gere 1 and the known cyclotide kalata B7 (kB7) were among the cyclotides characterized at the peptide and/or transcript level together with several putative enzymes, likely involved in cyclotide biosynthesis. Five of the most abundant cyclotides were isolated, sequenced, structurally characterized, and screened in antimicrobial and cytotoxicity assays. All gere cyclotides showed cytotoxicity (IC50 of 2.0-10.2 µM), but only gere 1 inhibited standard microbial strains at a minimum inhibitory concentration of 4-16 µM. As shown by immunohistochemistry, large quantities of the cyclotides were localized in the epidermis of the leaves and petioles of G. repens. Taken together with the cytotoxicity and membrane permeabilizing activities, this implicates gere cyclotides as potential plant defense molecules. The presence of cyO2-like gere 1 in a plant in the Rubiaceae supports the notion that phylogenetically distant plants may have coevolved to express similar cytotoxic cyclotides for a specific functional role, most likely involving host defense.

Rice and Arabidopsis BBX proteins: toward genetic engineering of abiotic stress resistant crops.

Productivity of crop plants are enormously affected by biotic and abiotic stresses. The co-occurrence of several abiotic stresses may lead to death of crop plants. Hence, it is the responsibility of plant scientists to develop crop plants equipped with multistress tolerance pathways. A subgroup of zinc finger transcription factor family, known as B-box (BBX) proteins, play a key role in light and hormonal regulation pathways. In addition, BBX proteins act as key regulatory proteins in many abiotic stress regulatory pathways, including Ultraviolet-B (UV-B), salinity, drought, heat and cold, and heavy metal stresses. Most of the BBX proteins identified in Arabidopsis and rice respond to more than one abiotic stress. Considering the requirement of improving rice for multistress tolerance, this review discusses functionally characterized Arabidopsis and rice BBX proteins in the development of abiotic stress responses. Furthermore, it highlights the participation of BBX proteins in multistress regulation and crop improvement through genetic engineering.

Good Practices in Sponge Natural Product Studies: Revising Vouchers with Isomalabaricane Triterpenes.

Species misidentification in the field of natural products is an acknowledged problem. These errors are especially widespread in sponge studies, albeit rarely assessed and documented. As a case study, we aim to revisit reports of isomalabaricane triterpenes, isolated from four demosponge genera: Jaspis, Geodia, Stelletta and Rhabdastrella. From a total of 44 articles (1981-2022), 27 unique vouchers were listed, 21 of which were accessed and re-examined here: 11 (52.4%) of these were misidentified. Overall, 65.9% of the studies published an incorrect species name: previously identified Jaspis and Stelletta species were all in fact Rhabdastrella globostellata. We conclude that isomalabaricane triterpenes were isolated from only two Rhabdastrella species and possibly one Geodia species. In addition to shedding a new light on the distribution of isomalabaricane triterpenes, this study is an opportunity to highlight the crucial importance of vouchers in natural product studies. Doing so, we discuss the impact of species misidentification and poor accessibility of vouchers in the field of sponge natural products. We advocate for stricter voucher guidelines in natural product journals and propose a common protocol of good practice, in the hope of reducing misidentifications in sponge studies, ensure reproducibility of studies, and facilitate follow-up work on the original material.

Tropical vibes from Sri Lanka - cyclotides from Viola betonicifolia by transcriptome and mass spectrometry analysis.

Cyclotides are an extremely stable class of peptides, ubiquitously distributed in Violaceae. The aim of the present study was to investigate the presence of cyclotides in Sri Lankan Violaceae plants, using combined tools of transcriptomics and mass spectrometry. New cyclotides were discovered for the first time in the wild flora of Sri Lanka, within Viola betonicifolia, a plant used in traditional medicine as an antimicrobial. Plant extracts prepared in small scale from Viola betonicifolia were first subjected to LC-MS analysis. Subsequent transcriptome de novo sequencing of Viola betonicifolia uncovered 25 new (vibe 1-25) and three known (varv A/kalata S, viba 17, viba 11) peptide sequences from Möbius and bracelet cyclotide subfamilies as well as hybrid cyclotides. Among the transcripts, putative linear acyclotide sequences (vibe 4, vibe 10, vibe 11 and vibe 22) that lack a conserved asparagine or aspartic acid vital for cyclisation were also present. Four asparagine endopeptidases (AEPs), VbAEP1-4 were found within the Viola betonicifolia transcriptome, including a peptide asparaginyl ligase (PAL), potentially involved in cyclotide backbone cyclisation, showing >93% sequence homology to Viola yedoensis peptide asparaginyl ligases, VyPALs. In addition, we identified two protein disulfide isomerases (PDIs), VbPDI1-2, likely involved in cyclotide oxidative folding, having high sequence homology (>74%) with previously reported Rubiaceae and Violaceae PDIs. The current study highlights the ubiquity of cyclotides in Violaceae as well as the utility of transcriptomic analysis for cyclotides and their putative processing enzyme discovery. The high variability of cyclotide sequences in terms of loop sizes and residues in V. betonicifolia showcase the cyclotide structure as an adaptable scaffold as well as their importance as a combinatorial library, implicated in plant defense.

Molecular characterization and expression of α-gliadin genes from wheat cultivar Dacke in Bg 250 rice variety.

The main seed storage protein in wheat is gluten. It consists of gliadin and glutenins. Gluten gives high elasticity and extensibility during bread making, facilitating the formation of the dough. Rice is the staple food of Sri Lankans but, it has poor dough making ability compared to wheat. The aim of the present work was to characterize, clone and express α-gliadin in the T0 generation of Bg 250 rice variety as a preliminary step in improving the dough making ability of rice flour. Five α-gliadin recombinant pCR™2.1-TOPO® clones were selected for sequence analysis. Of the five clones, two functional genes and three pseudogenes were identified. Phylogenetic analysis revealed the two functional genes, (accession numbers KC660359 and KC660358) to be closely related to the α-gliadin genes of Triticum monococcum. The α-gliadin gene (KC660359) contained five cysteine residues, one less than the normal occurrence of cysteine residues in α-gliadin genes. To date there are no reports on expression of gliadin gene in transgenic rice. This novel gene was successfully expressed in the Sri Lankan rice variety Bg 250 under the control of the rice GluB-1 endosperm specific promoter.

Arabidopsis COP1 SUPPRESSOR 2 Represses COP1 E3 Ubiquitin Ligase Activity through Their Coiled-Coil Domains Association.

CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) functions as an E3 ubiquitin ligase and mediates a variety of developmental processes in Arabidopsis by targeting a number of key regulators for ubiquitination and degradation. Here, we identify a novel COP1 interacting protein, COP1 SUPPRESSOR 2 (CSU2). Loss of function mutations in CSU2 suppress the constitutive photomorphogenic phenotype of cop1-6 in darkness. CSU2 directly interacts with COP1 via their coiled-coil domains and is recruited by COP1 into nuclear speckles in living plant cells. Furthermore, CSU2 inhibits COP1 E3 ubiquitin ligase activity in vitro, and represses COP1 mediated turnover of HY5 in cell-free extracts. We propose that in csu2 cop1-6 mutants, the lack of CSU2's repression of COP1 allows the low level of COP1 to exhibit higher activity that is sufficient to prevent accumulation of HY5 in the dark, thus restoring the etiolated phenotype. In addition, CSU2 is required for primary root development under normal light growth condition.

The RING-Finger E3 Ubiquitin Ligase COP1 SUPPRESSOR1 Negatively Regulates COP1 Abundance in Maintaining COP1 Homeostasis in Dark-Grown Arabidopsis Seedlings.

CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) functions as an E3 ubiquitin ligase in both plants and animals. In dark-grown Arabidopsis thaliana seedlings, COP1 targets photomorphogenesis-promoting factors for degradation to repress photomorphogenesis. Little is known, however, about how COP1 itself is regulated. Here, we identify COP1 SUPPRESSOR1 (CSU1), a RING-finger E3 ubiquitin ligase, as a regulator of COP1. Genetic evidence demonstrates that csu1 mutations suppress cop1-6 phenotypes completely in the dark. Furthermore, CSU1 colocalizes with COP1 in nuclear speckles and negatively regulates COP1 protein accumulation in darkness. CSU1 can ubiquitinate COP1 in vitro and is essential for COP1 ubiquitination in vivo. Therefore, we conclude that CSU1 plays a major role in maintaining COP1 homeostasis by targeting COP1 for ubiquitination and degradation in dark-grown seedlings.

Convergence of Light and ABA signaling on the ABI5 promoter.

Light is one of the most important environmental cues regulating multiple aspects of plant growth and development, and abscisic acid (ABA) is a plant hormone that plays important roles during many phases of the plant life cycle and in plants' responses to various environmental stresses. How plants integrate the external light signal with endogenous ABA pathway for better adaptation and survival remains poorly understood. Here, we show that BBX21 (also known as SALT TOLERANCE HOMOLOG 2), a B-box (BBX) protein previously shown to positively regulate seedling photomorphogenesis, is also involved in ABA signaling. Our genetic data show that BBX21 may act upstream of several ABA INSENSITIVE (ABI) genes and ELONGATED HYPOCOTYL 5 (HY5) in ABA control of seed germination. Previous studies showed that HY5 acts as a direct activator of ABI5 expression, and that BBX21 interacts with HY5. We further demonstrate that BBX21 negatively regulates ABI5 expression by interfering with HY5 binding to the ABI5 promoter. In addition, ABI5 was shown to directly activate its own expression, whereas BBX21 negatively regulates this activity by directly interacting with ABI5. Together, our study indicates that BBX21 coordinates with HY5 and ABI5 on the ABI5 promoter and that these transcriptional regulators work in concert to integrate light and ABA signaling in Arabidopsis thaliana.

The Arabidopsis B-BOX protein BBX25 interacts with HY5, negatively regulating BBX22 expression to suppress seedling photomorphogenesis.

ELONGATED HYPOCOTYL5 (HY5) is a basic domain/leucine zipper (bZIP) transcription factor, central for the regulation of seedling photomorphogenesis. Here, we identified a B-BOX (BBX)-containing protein, BBX25/SALT TOLERANCE HOMOLOG, as an interacting partner of HY5, which has been previously found to physically interact with CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1). BBX25 physically interacts with HY5 both in vitro and in vivo. By physiological and genetic approaches, we showed that BBX25 is a negative regulator of seedling photomorphogenesis. BBX25 and its homolog BBX24 regulate deetiolation processes and hypocotyl shade avoidance response in an additive manner. Moreover, genetic relationships of bbx25 and bbx24 with hy5 and cop1 revealed that BBX25 and BBX24 additively enhance COP1 and suppress HY5 functions. BBX25 accumulates in a light-dependent manner and undergoes COP1-mediated degradation in dark and light conditions. Furthermore, a protoplast cotransfection assay showed that BBX24 and BBX25 repress BBX22 expression by interfering with HY5 transcriptional activity. As HY5 binds to the BBX22 promoter and promotes its expression, our results identify a direct mechanism through which the expression of BBX22 is regulated. We suggest that BBX25 and BBX24 function as transcriptional corepressors, probably by forming inactive heterodimers with HY5, downregulating BBX22 expression for the fine-tuning of light-mediated seedling development.

In Vitro Antibacterial Activity of 4-Phenyl-1-(2-phenyl-allyl)pyridinium bromide: A Novel Class of Pyridinium Based Antibacterial Compounds.

The continuing increase in the incidence of multi drug resistant pathogenic bacteria and shortage of new antimicrobial agents are the prime driver in efforts to identify the novel antimicrobial classes. In vitro antibacterial activity of 4-phenyl-1-(2-phenylallyl) pyridinium bromide was tested against Gram positive Staphylococcus aureus, Streptococcus species, Bacillus subtilis, and Gram negative Klebsiella aerogenes and Escherichia coli using disk diffusion method. Among them S. aureus showed strong antibacterial activity (21.99 ± 0.03 mm) while E. coli showed very little activity (8.97 ± 0.06 mm) towards the compound. The MIC of 4-phenyl-1-(2-phenyl-allyl)-pyridinium bromide for 90% S. aureus was ≤20 µg/ml and was compared with phenoxymethylpenicillin, cloxacillin, erythromycin and vancomycin. When 4-phenyl-1-(2-phenyl-allyl)pyridinium bromide showed MIC at ≤20 µg/ml, all others showed MIC at ≤100 µg/ml. Strong antibacterial activity of 4-phenyl-1-(2-phenyl-allyl)pyridinium bromide against S. aureus indicates that there is a possibility to use it as an effective antibacterial agent.

LZF1/SALT TOLERANCE HOMOLOG3, an Arabidopsis B-box protein involved in light-dependent development and gene expression, undergoes COP1-mediated ubiquitination.

B-box containing proteins play an important role in light signaling in plants. Here, we identify LIGHT-REGULATED ZINC FINGER1/SALT TOLERANCE HOMOLOG3 (STH3), a B-box encoding gene that genetically interacts with two key regulators of light signaling, ELONGATED HYPOCOTYL5 (HY5) and CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1). STH3 physically interacts with HY5 in vivo and shows a COP1-dependent localization to nuclear speckles when coexpressed with COP1 in plant cells. A T-DNA insertion mutant, sth3, is hyposensitive to high fluence blue, red, and far-red light and has elongated hypocotyls under short days. Analyses of double mutants between sth3, sth2, and hy5 suggest that they have partially overlapping functions. Interestingly, functional assays in protoplasts suggest that STH3 can activate transcription both independently and together with STH2 through the G-box promoter element. Furthermore, sth3 suppresses the cop1 hypocotyl phenotype in the dark as well as the anthocyanin accumulation in the light. Finally, COP1 ubiquitinates STH3 in vitro, suggesting that STH3 is regulated by COP1. In conclusion, we have identified STH3 as a positive regulator of photomorphogenesis acting in concert with STH2 and HY5, while also being a target of COP1-mediated ubiquitination.

STH2 has 2 B there: An insight into the role of B-box containing proteins in Arabidopsis.

Recently we reported the identification of a novel B-box transcription factor SALT TOLERANCE HOMOLOG 2 (STH2) that interacts genetically with two key regulators of the light-signaling pathway, HY5 and COP1.1 We also provided phenotypic and genetic characterization of the sth2 mutant suggesting that STH2 plays a positive role in regulating photomorphogenesis both independently or together with HY5. Functional assays in protoplasts revealed that STH2 could act as a transcriptional activator. To our knowledge this is the first report of a B-box domain containing protein playing a direct role in activating transcription in plants. Here we discuss the possible position of STH2 in the transcriptional network and comment on the role of the B-box domain in plants.

SALT TOLERANCE HOMOLOG2, a B-box protein in Arabidopsis that activates transcription and positively regulates light-mediated development.

CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1) and ELONGATED HYPOCOTYL5 (HY5) are two major regulators of light signaling in plants. Here, we identify SALT TOLERANCE HOMOLOG2 (STH2) as a gene that interacts genetically with both of these key regulators. STH2 encodes a B-box-containing protein that interacts physically with HY5 in yeast and in plant cells. Whereas STH2 is uniformly nuclear by itself, it shows a COP1-dependent localization to speckles when coexpressed with COP1. We identified two independent T-DNA insertion lines in STH2. Both alleles are hyposensitive to blue, red, and far-red light. The sth2 mutant, like hy5, shows an enhanced number of lateral roots and accumulates less anthocyanin. Analysis of double mutants between sth2 and hy5 indicates that STH2 has both HY5-dependent and -independent functions. Furthermore, besides partially suppressing the hypocotyl phenotype of dark-grown cop1 alleles, sth2 also suppresses the reduced number of lateral roots and high anthocyanin levels in light-grown cop1 alleles. Interestingly, we found that STH2 can activate transcription. Transient transfection assays in protoplasts using a LUC reporter driven by the chalcone isomerase promoter show that the B-boxes in STH2 and a functional G-box element in the promoter are required for this activity. In conclusion, we have identified STH2, a B-box protein in Arabidopsis thaliana, as a positive regulator of photomorphogenesis and report that the B-box domain plays a direct role in activating transcription in plants.

Opposite root growth phenotypes of hy5 versus hy5 hyh mutants correlate with increased constitutive auxin signaling.

The Arabidopsis transcription factor HY5 controls light-induced gene expression downstream of photoreceptors and plays an important role in the switch of seedling shoots from dark-adapted to light-adapted development. In addition, HY5 has been implicated in plant hormone signaling, accounting for the accelerated root system growth phenotype of hy5 mutants. Mutants in the close HY5 homolog HYH resemble wild-type, despite the largely similar expression patterns and levels of HY5 and HYH, and the functional equivalence of the respective proteins. Moreover, the relative contribution of HYH to the overall activity of the gene pair is increased by an alternative HYH transcript, which encodes a stabilized protein. Consistent with the enhanced root system growth observed in hy5 loss-of-function mutants, constitutively overexpressed alternative HYH inhibits root system growth. Paradoxically, however, in double mutants carrying hy5 and hyh null alleles, the hy5 root growth phenotype is suppressed rather than enhanced. Even more surprisingly, compared to wild-type, root system growth is diminished in hy5 hyh double mutants. In addition, the double mutants display novel shoot phenotypes that are absent from either single mutant. These include cotyledon fusions and defective vasculature, which are typical for mutants in genes involved in the transcriptional response to the plant hormone auxin. Indeed, many auxin-responsive and auxin signaling genes are misexpressed in hy5 mutants, and at a higher number and magnitude in hy5 hyh mutants. Therefore, auxin-induced transcription is constitutively activated at different levels in the two mutant backgrounds. Our data support the hypothesis that the opposite root system phenotypes of hy5 single and hy5 hyh double mutants represent the morphological response to a quantitative gradient in the same molecular process, that is gradually increased constitutive auxin signaling. The data also suggest that HY5 and HYH are important negative regulators of auxin signaling amplitude in embryogenesis and seedling development.