Functional differences between two major ubiquitin receptors in the proteasome; S5a and hRpn13

It is well known that S5a and hRpn13 are two major ubiquitin (Ub) receptors in the proteasome but little is known about their functional difference in recruiting ubiquitinated substrates. In this study using siRNA-mediated knockdown of S5a or hRpn13, we found that two Ub receptors had different substrate specificity although similar level of accumulation of high molecular weight Ub-conjugates was observed. Interesting enough, depletion of S5a, but not hRpn13, resulted in the Ub-containing aggregates and induced ER chaperones such as Grp78 and Grp94. ERAD substrates such as α-TCR and α1-antitrypsin were also stabilized by the depletion of S5a but not hRpn13. Our results suggest that there is different substrate specificity between S5a and hRpn13 at the level of delivery and S5a may be the major docking site for ERAD substrates.     

Induction of systemic resistance by mixtures of antagonist bacteria for the management of crown rot complex on banana

Among nine native bacterial strains isolated from banana fruit surface and rhizosphere and six bacterial strains introduced from the culture collection, three native strains viz., non-fluorescent Pseudomonas (NFP₆), Pseudomonas fluorescens (Pf3a), and Bacillus subtilis (BS₁); and two bacterial strains from culture collection viz., Azospirillum (AS1) and Azotobacter (AZ1) have recorded maximum inhibition of mycelial growth of crown rot pathogens (Lasiodiplodia theobromae and Colletotrichum musae) under in vitro condition. When these effective bacterial strains were treated on banana fruits under in vivo, significant reduction of crown rot disease and increased shelf life of banana was observed. However, bacterial strains applied as three way combinations (NFP₆ + Pf3a + BS₁) had greater effect compared with individual and two way combination of bacterial antagonist treatments. The effect of crown rot disease reduction was also comparable to that of fungicide Benomyl (0.1%) both under cold and room temperature storage conditions. Besides, the induction of defense-related enzymes such as phenylalanine ammonia-lyase (PAL), peroxidase (PO), polyphenoloxidase (PPO), and the accumulation of phenolics in banana fruit due to the application of bacterial antagonists were also studied at five different time intervals viz. 0th, 1st, 3rd, 5th and 7th days after treatment. When banana fruits treated with bacterial antagonists (individually and also in different combinations) and challenge-inoculated with crown rot pathogens, up to fourfold increase in defense-related enzymes and 3.6 fold increase in phenolic content was observed compared with control. The activity of these defense-related enzymes and phenolic content had gradually increased from 1st day after treatment to 3rd after treatment and reached their peak on 5th day after treatment. Among the bacterial antagonists which have been applied individually and in different combinations, the banana fruits treated with three-way antagonist mixture, i.e., NFP₆ + Pf3a + BS₁ recorded maximum induction of defense-related enzymes and accumulation of phenolics compared with individual and two-way combination of antagonist mixtures. This study suggest that the increased induction of defense-related enzymes and phenolic content due to the treatment of banana fruits with bacterial antagonists might have involved in the reduction of crown rot severity and in turn increased the shelf life of banana fruits.     

Purification and characterization of laccase produced by a white rot fungus Pleurotus sajor-caju under submerged culture condition and its potential in decolorization of azo dyes

An extracellular laccase was isolated and purified from Pleurotus sajor-caju grown in submerged culture in a bioreactor, and used to investigate its ability to decolorize three azo dyes. The extracellular laccase production was enhanced up to 2.5-fold in the medium amended with xylidine (1 mM). Purification was carried out using ammonium sulfate (70% w/v), DEAE-cellulose, and Sephadex G-100 column chromatography. The enzyme was purified up to 10.3-fold from the initial protein preparation with an overall yield of 53%. The purified laccase was monomeric with an apparent molecular mass of 61.0 kDa. The purified enzyme exerted its optimal activity with 2,2-azino–bis(3-ethylbenzo-thiazoline-6-sulfonate (ABTS) and oxidized various lignin-related phenols. The catalytic efficiencies k _cat/K _m determined for ABTS and syringaldazine were 9.2×10⁵ and 8.7×10⁵, respectively. The optimum pH and temperature for the purified enzyme was 5.0 and 40 °C, respectively. Sodium azide completely inhibited the laccase activity. The absorption spectrum revealed type 1 and type 3 copper signals. The purified enzyme decolorized azo dyes such as acid red 18, acid Black 1, and direct blue 71 up to 90, 87, and 72%, respectively. Decolorization ability of P. sajor-caju laccase suggests that this enzyme could be used for decolorization of industrial effluents.     

Langevin dynamics simulations of genome packing in bacteriophage

We use Langevin dynamics simulations to study the process by which a coarse-grained DNA chain is packaged within an icosahedral container. We focus our inquiry on three areas of interest in viral packing: the evolving structure of the packaged DNA condensate; the packing velocity; and the internal buildup of energy and resultant forces. Each of these areas has been studied experimentally, and we find that we can qualitatively reproduce experimental results. However, our findings also suggest that the phage genome packing process is fundamentally different than that suggested by the inverse spool model. We suggest that packing in general does not proceed in the deterministic fashion of the inverse-spool model, but rather is stochastic in character. As the chain configuration becomes compressed within the capsid, the structure, energy, and packing velocity all become dependent upon polymer dynamics. That many observed features of the packing process are rooted in condensed-phase polymer dynamics suggests that statistical mechanics, rather than mechanics, should serve as the proper theoretical basis for genome packing. Finally we suggest that, as a result of an internal protein unique to bacteriophage T7, the T7 genome may be significantly more ordered than is true for bacteriophage in general.     

Arbuscular mycorrhizal and dark septate endophyte fungal associations in South Indian grasses

We examined arbuscular mycorrhizal (AM) and dark septate endophyte (DSE) fungal association in 50 south Indian grasses from four different sites. AM fungal diversity was also compared among the different sites. Forty-four of the 50 grasses examined had AM association and dual association with DSE fungi occurred in 25 grasses. We report for the first time AM and DSE fungal status in 23 and 27 grasses respectively. Arum-type AM morphology was the dominant occurring in 21 grasses with typical Paris-type colonization occurring in 6 grasses. AM morphology is reported for the first time in 35 grasses. Over the different sites, spore density in the soil ranged from 5–22 per 100 g air-dried soil. Spores of 11 AM fungal taxa were isolated from the soil samples of grasses of which nine belonged to Glomus, one to Acaulospora and one to Scutellospora. No significant relationship existed between AM fungal colonization and spore numbers. Species richness was high in site II and Glomus aggregatum, Glomus viscosum and Glomus mosseae were most frequent species at different sites. Overall species diversity indices (Simpson index, Shannon-Weaver index, species equitability index) differed significantly between sites.     

Length-weight relationships of eight elasmobranch species captured along the Coromandel coast of Tamil Nadu,Eastern Indian Ocean

This study reports the length-weight relationships (LWR) for eight elasmobranch species; four shark species (Carcharhiniformes and Orectolobiformes), two species of rays (Myliobatiformes) and two species of guitarfishes (Rhinopristiformes) from the Coromandel Coast of Tamil Nadu, Eastern Indian Ocean. The specimens of these elasmobranch species were collected from the catches of various gears like trawl net, bottom set and drift gill net, hook & line at Chennai—Royapuram Fishing Harbour, Cuddalore Fishing Harbourand Nagapattinam Fishing Harbour situated along this coastal region fortnightly during June 2019–March 2020. The values of the parameter ‘b’ remained within the expected range of 2.5–3.5. Length- weight / Disc-width-weight relationships showed good fit with r² values varying from 0.8923 to 0.9869. This study also reports a new maximum TL length (L_max)for a shark species (Chiloscyllium griseum).     

Boranophosphate Oligonucleotides: New Synthetic Approaches

Abstract

Recently our laboratory reported a new backbone-modified class of oligonucleotides, with a borane (B₃3?) group replacing one of the non-bridging oxygen atoms. Here we present two new approaches to synthesize the boranophosphate oligonucleotides. All-stereoregular boranophosphate oligonucleotides can be prepared by enzymatic template extension reactions using nucleoside a-boranotriphosphates, which are good substrates for a number of polymerases. Larger scale synthesis of boranophosphate oligonucleotides can be carried out by effective chemical synthesis using the H-phosphonate approach, instead of previously used phosphoramidite methodology. The main advantage of H-phosphonate methodology is the ability to carry out one boronation reaction, after oligonucleotide chain elongation has been completed, using mild conditions without base damage and producing the desired boranophosphate oligonucleotides in high yield.     

Stable Transformation of Ferns Using Spores as Targets: Pteris vittata and Ceratopteris thalictroides

Ferns (Pteridophyta) are very important members of the plant kingdom that lag behind other taxa with regards to our understanding of their genetics, genomics, and molecular biology. We report here, to our knowledge, the first instance of stable transformation of fern with recovery of transgenic sporophytes. Spores of the arsenic hyperaccumulating fern Pteris vittata and tetraploid ‘C-fern Express’ (Ceratopteris thalictroides) were stably transformed by Agrobacterium tumefaciens with constructs containing the P. vittata actin promoter driving a GUSPlus reporter gene. Reporter gene expression assays were performed on multiple tissues and growth stages of gametophytes and sporophytes. Southern-blot analysis confirmed stable transgene integration in recovered sporophytes and also confirmed that no plasmid from A. tumefaciens was present in the sporophyte tissues. We recovered seven independent transformants of P. vittata and four independent C. thalictroides transgenics. Inheritance analyses using β-glucuronidase (GUS) histochemical staining revealed that the GUS transgene was stably expressed in second generation C. thalictroides sporophytic tissues. In an independent experiment, the gusA gene that was driven by the 2× Cauliflower mosaic virus 35S promoter was bombarded into P. vittata spores using biolistics, in which putatively stable transgenic gametophytes were recovered. Transformation procedures required no tissue culture or selectable marker genes. However, we did attempt to use hygromycin selection, which was ineffective for recovering transgenic ferns. This simple stable transformation method should help facilitate functional genomics studies in ferns.Ferns (Pteridophyta) are nonflowering vascular plants comprised of 250 genera, the second largest group of diversified species in the plant kingdom (Gifford and Foster, 1989). Many interesting traits are inherent in various fern species, such as arsenic hyperaccumulation (Pteris vittata; Ma et al., 2001), insecticide production and allelopathy (Pteridium aquilinum; Marrs and Watt, 2006), and antimicrobial compound production (Acrostichum aureum; Lai et al., 2009). Ferns occupy the evolutionary niche between nonvascular land plants, bryophytes, and higher vascular plants such as gymnosperms and angiosperms. Therefore, extant fern species still hold a living record of the initial adaptations required for plants to thrive on land. Of these adaptations, the most important is tracheids that comprise xylem tissues for water and mineral transport and structural support. The vascular system allowed pteridophytes to grow upright during the sporophyte generation, leading to greater resource acquisition capacity. Ultimately, sufficient resources allowed for greater spore production and upright growth, which facilitated spore spread. Recent endeavors have investigated lower plant genomics, including the sequencing of the bryophyte Physcomitrella patens (Rensing et al., 2008) and the lycophyte Selaginella moellendorffii (Banks et al., 2011). Both basic and applied biology of ferns lag far behind that for angiosperms and even bryophytes.Stable genetic transformation has been accomplished in a few species outside angiosperms and gymnosperms, especially among bryophytes (Schaefer et al., 1991; Chiyoda et al., 2008; Ishizaki et al., 2008), but never in the Pteridophyta. Transient transformation methods have been developed both in Ceratopteris richardii ‘C-fern’ and P. vittata, which were limited to heterologous expression in gametophytes (Rutherford et al., 2004; Indriolo et al., 2010). While transient expression of transgenic constructs does enable research, there is no substitute for stable transformation in functional genomics and plant biology. Researchers have resorted to studying fern gene function using heterologous expression in the angiosperm model plant Arabidopsis (Arabidopsis thaliana; Dhankher et al., 2006; Sundaram et al., 2009; Sundaram and Rathinasabapathi, 2010), which is far from optimal. Overexpression and knockdown analysis of individual genes in a wide variety of fern species would undoubtedly accelerate our ability to learn more about their biology and subsequently develop novel products from ferns. Furthermore, a facile transformation system would accelerate functional genomics and systems biology of ferns. For example, knockdown analysis of genes involved in interesting biosynthetic pathways can greatly facilitate gene and biochemical discovery.P. vittata has the unparalleled ability to accumulate more arsenic per gram biomass than any other plant species and is highly tolerant to arsenic (Gumaelius et al., 2004). It can thrive in soils containing up to 1,500 µg mL^–1 arsenic, whereas most plants cannot survive 50 µg mL^–1 arsenic (Ma et al., 2001). Therefore, P. vittata has been the subject of extensive basic and applied research for arsenic hyperaccumulation, translocation, and resistance (Gumaelius et al., 2004) and has been used for arsenic phytoremediation (Shelmerdine et al., 2009). For example, this fern might be of great utility for the production of a safer rice (Oryza sativa) crop; arsenic can be transported and stored in the grain, resulting in serious human health ramifications (Srivastava et al., 2012). In a recent greenhouse study, P. vittata has been used to remediate arsenic-contaminated soil. Following remediation, rice plants were subsequently grown, and it was found that arsenic uptake by rice grains was reduced by 52%, resulting in less than 1 µg mL^–1 arsenic after two rounds of remediation using P. vittata phytoremediation (Mandal et al., 2012). Furthermore, this treatment also resulted in increased rice grain yield by 14% (w/w) compared with control.Ceratopteris is a subtropical-to-tropical fern genus containing four to six species living in aquatic habitats. The C-fern cultivar was developed as a model fern for teaching (http://www.c-fern.org) and research owing to its small size, short life cycle (120 d), and its amenability for in vitro culture (Banks, 1999). The C-fern Express cultivar was developed by Leslie G. Hickok by crossing two Japanese varieties of Ceratopteris thalictroides (L. Hickok, personal communication). cv C-fern Express, a tetraploid, develops spores in 60 d of culture. Though cv C-fern spores have been shown to be a useful single cell model system and a rapid and efficient system for studying RNA interference in ferns (Stout et al., 2003), no stable transformation studies have been reported. In bryophytes, immature thalli are most often used as explants (Chiyoda et al., 2008; Ishizaki et al., 2008), whereas in fungi, spores are routinely used for stable transformation studies (Michielse et al., 2005; Utermark and Karlovsky, 2008).The objective of our research was to develop, for the first time, a facile stable transformation system for P. vittata and C. thalictroides using spores as the transformation targets. This method can be used as an additional tool to further substantiate and strengthen the molecular mechanism studies in pteridophytes.     

Asymbiotic seed germination of Cymbidium bicolor Lindl. (Orchidaceae) and the influence of mycorrhizal fungus on seedling development

An in vitro plant regeneration protocol was successfully established for Cymbidium bicolor an epiphytic orchid by culturing seeds from green pods. Immature seeds were germinated on four basal media viz., Murashige and Skoog (MS) medium, Knudson C (KC) orchid medium, Knudson C modified Morel (KCM) medium and Lindemann orchid (LO) medium. Seed germination and protocorm development was significantly higher in LO medium (96.6 %) followed by KCM (89 %), MS (77.5 %) and KC (62.7 %) media after 56 days. For multiple shoot induction the protocorms were transferred to B5 medium supplemented with cytokinin. Among the various cytokinins tested, BAP (4.42 μM) induced maximum (27.59) number of multiple shoots per explant. IBA was effective in inducing healthy roots. Tissue-cultured protocorms and seedlings of C. bicolor were inoculated with AC-01 fungal strain (Moniliopsis sp.) isolated from the mycorrizal roots of an epiphytic orchid Aerides crispum. Mycorrhizal fungi significantly enhanced number of roots, root length and shoot number.