ANTIOXIDANT, ANTIBACTERIAL AND ANTI-INFLAMMATORY POTENTIAL OF THE AQUEOUS EXTRACT OF THE RAW LEAVES OF SRI LANKAN VARIETY OF PERSEA AMERICANA MILLER (AVOCADO)

Despite the advances in modern medicine, plant based remedies are currently sought after as alternatives to synthetic drugs. As such natural sources are being searched to use as antioxidants, anti-inflammatory, and antibacterial compounds. This study was focused on the investigation of aqueous leaf extract of Sri Lankan variety of Persea americana (ALEPA) which was prepared according to the method of Kasaya (Decoction) in Ayurvedic medicine to test its antioxidant, anti-inflammatory and antibacterial activities. The total phenolic content of ALEPA was determined by Folin Ciocalteu assay was 681±6.8 mg (PGE) /g (PGE= Pyrogallol equivalents) and the flavonoid content was 1193±302 mg (QE)/g (QE= Quercetin equivalents) according to the AlCl3 colorimetric assay. DPPH radical scavenging activity (RSA) of ALEPA was 11.3±2.8-52.3±5.0% for concentrations of 50-500 µg/ml whereas ascorbic acid showed 28.0±0.7-90.7±0.1% in the concentration range of 200-500 µg/ml. ALEPA showed better ferrous ion reducing activity compared to ascorbic acid. Hydroxyl RSA of ALEPA was 17.4±1.3-41.5±0.5% in the concentration range of 20-50mg/ml whereas ascorbic acid showed 7.0±0.2-19.8±0.5% for the same concentration range. Overproduction of nitric oxide free radicals are implicated in inflammation. In the NO scavenging assay, ALEPA showed activity 26.0±3.6-69.2±2.5% in the concentrations of 0.2-8 ppm whereas ascorbic acid showed activity 26.8±2.5-49.3±1.9% in the concentrations of 0.2-4 ppm. In human red blood cell (HRBC) assay, ALEPA showed 48.0±1.9-60.6±2.6% HRBC membrane protection whereas the Asprin showed 39.7±0.4-49.0±0.5% activity. The prevention of HRBC membrane lysis is taken as a measure of anti-inflammatory activity and also ALEPA showed antibacterial activity towards Escherichia coli, Streptococcus aureus, and Bacillus subtilis

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.Fil: Gangappa, Sreeramaiah N.. Gothenburg University. Department of Biological and Environmental Sciences; SueciaFil: Crocco, Carlos Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Fisiológicas y Ecológicas Vinculadas A la Agricultura; ArgentinaFil: Johansson, Henrik. Gothenburg University. Department of Biological and Environmental Sciences; SueciaFil: Datta, Sourav. Gothenburg University. Department of Biological and Environmental Sciences; SueciaFil: Hettiarachchi, Chamari. Gothenburg University. Department of Biological and Environmental Sciences; SueciaFil: Holm, Magnus. Gothenburg University. Department of Biological and Environmental Sciences; SueciaFil: Botto, Javier Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Fisiológicas y Ecológicas Vinculadas A la Agricultura; Argentin

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

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

SALT TOLERANCE HOMOLOG2, a B-Box Protein in Arabidopsis That Activates Transcription and Positively Regulates Light-Mediated Development[W]

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

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 Mobius 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

LZF1/SALT TOLERANCE HOMOLOG3, an Arabidopsis B-Box Protein Involved in Light-Dependent Development and Gene Expression, Undergoes COP1-Mediated Ubiquitination[W]

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