Biotechnological advances in guava (Psidium guajava L.): recent developments and prospects for further research

Guava (Psidium guajava L.), an important fruit crop of several tropical and sub-tropical countries, is facing several agronomic and horticultural problems such as susceptibility to many pathogens, particularly guava wilting caused by Fusarium oxysporium psidii, low fruit growth, short shelf life of fruits, high seed content, and stress sensitivity. Conventional breeding techniques have limited scope in improvement of guava owing to long juvenile period, self incompatibility, and heterozygous nature. Conventional propagation methods, i.e., cutting, grafting or stool layering, for improvement of guava already exist, but the long juvenile period has made them time consuming and cumbersome. Several biotechnological approaches such as genetic transformation may be effective practical solutions for such problems and improvement of guava. The improvement of fruit trees through genetic transformation requires an efficient regeneration system. During the past 2–3 decades, different approaches have been made for in vitro propagation of guava. An overview on the in vitro regeneration of guava via organogenesis, somatic embryogenesis, and synthetic seeds is presented. Organogenesis in several different genotypes through various explant selection from mature tree and seedling plants has been achieved. Factors affecting somatic embryogenesis in guava have been reviewed. Production of synthetic seeds using embryogenic propagules, i.e., somatic embryos and non-embryogenic vegetative propagules, i.e., shoot tips and nodal segments have also been achieved. Development of synthetic seed in guava may be applicable for propagation, short-term storage, and germplasm exchange, and distribution. An initial attempt for genetic transformation has also been reported. The purpose of this review is to focus upon the current information on in vitro propagation and biotechnological advances made in guava.     

Alginate-encapsulation of nodal segments for propagation, short-term conservation and germplasm exchange and distribution of Eclipta alba (L.)

Nodal segments obtained from in vitro proliferated shoots of Eclipta alba (L.) Hassk, were encapsulated in calcium alginate beads for large-scale clonal propagation, short-term conservation and germplasm exchange and distribution. The best gel complexation was achieved using 3% sodium alginate and 100 mM CaCl₂·2H₂O. Maximum percent response (100%) for conversion of encapsulated nodal segments into plantlets was obtained on 0.7% agar-solidified full-strength MS medium containing 0.88 μM BAP. Encapsulated nodal segments could be stored at low temperature (4°C) up to 60 days with a survival frequency of 51.2%. The well-developed plantlets regenerated from encapsulated nodal segments were hardened-off successfully with 90% survival frequency.     

Study on the effects of chloride depletion on photosystem II using different chloride depletion methods

Chloride is an indispensable factor for the functioning of oxygen evolving complex (OEC) and has protective and activating effects on photosystem II. In this study we have investigated mainly by EPR, the properties of chloride-sufficient, chloride-deficient and chloride-depleted thylakoid membranes and photosystem II enriched membranes from spinach. The results on the effects of different chloride depletion methods on the structural and functional aspects of photosystem II showed that chloride-depletion by treating PS II membranes with high pH is a relatively harsh way causing a significant and irreparable damage to the PS II donor side. Damage to the acceptor side of PS II was recovered almost fully in chloride-deficient as well as chloride-depleted PS II membranes.     

The Pneumococcal Serine-Rich Repeat Protein Is an Intra-Species Bacterial Adhesin That Promotes Bacterial Aggregation In Vivo and in Biofilms

The Pneumococcal serine-rich repeat protein (PsrP) is a pathogenicity island encoded adhesin that has been positively correlated with the ability of Streptococcus pneumoniae to cause invasive disease. Previous studies have shown that PsrP mediates bacterial attachment to Keratin 10 (K10) on the surface of lung cells through amino acids 273–341 located in the Basic Region (BR) domain. In this study we determined that the BR domain of PsrP also mediates an intra-species interaction that promotes the formation of large bacterial aggregates in the nasopharynx and lungs of infected mice as well as in continuous flow-through models of mature biofilms. Using numerous methods, including complementation of mutants with BR domain deficient constructs, fluorescent microscopy with Cy3-labeled recombinant (r)BR, Far Western blotting of bacterial lysates, co-immunoprecipitation with rBR, and growth of biofilms in the presence of antibodies and competitive peptides, we determined that the BR domain, in particular amino acids 122–166 of PsrP, promoted bacterial aggregation and that antibodies against the BR domain were neutralizing. Using similar methodologies, we also determined that SraP and GspB, the Serine-rich repeat proteins (SRRPs) of Staphylococcus aureus and Streptococcus gordonii, respectively, also promoted bacterial aggregation and that their Non-repeat domains bound to their respective SRRPs. This is the first report to show the presence of biofilm-like structures in the lungs of animals infected with S. pneumoniae and show that SRRPs have dual roles as host and bacterial adhesins. These studies suggest that recombinant Non-repeat domains of SRRPs (i.e. BR for S. pneumoniae) may be useful as vaccine antigens to protect against Gram-positive bacteria that cause infection.     

Presence of two types of flowers with respect to nectar sugar in two gregariously flowering species

Many species of animal-pollinated flowers are known to vary widely in the nectar content of flowers. Some proportion of flowers in many species is apparently nectarless, and such flowers are believed to be ‘cheaters’. Cheating may explain a part of the variability in nectar content. If cheating exists as a qualitatively different strategy then we expect bimodality in the distribution of nectar content of flowers. It has been shown in a multispecies study that gregarious species have a higher proportion of cheater flowers. We studied the frequency distribution of total nectar sugar in two gregariously flowering species Lantana camara and Utricularia purpurascens, which differed in other floral and ecological characters. At the population level, both the species showed significant bimodality in the total sugar content of flowers. The obvious sources of heterogeneity in the data did not explain bimodality. In Lantana camara, bimodality was observed within flowers of some of the individual plants sampled. In Utricularia purpurascens the proportion of nectarless flowers was more in high-density patches, suggesting that the gregariousness hypothesis may work within a species as well. The results support the hypothesis of cheating as a distinct strategy since two distinct types of flowers were observed in both the species. The effect of density in Utricularia purpurascens also supports the gregariousness hypothesis.     

Trifunctional norrisolide probes for the study of Golgi vesiculation

Inspired by the effect of norrisolide on the Golgi complex, we synthesized norrisolide probes that contain: the perhydroindane core of the parent natural product for Golgi localization, a crosslinking unit (aryl azide or epoxide) for covalent binding to the target, and a tag (biotin or iodine) for subsequent target purification. We found that biotin-containing probes 14, 20 and 24 induced inefficient Golgi vesiculation. However, the iodinated probe 25 induced extensive and irreversible Golgi fragmentation. This probe can be used for the isolation of the cellular target of norrisolide.     

Crystal structure of RNase T, an exoribonuclease involved in tRNA maturation and end turnover

The 3' processing of most bacterial precursor tRNAs involves exonucleolytic trimming to yield a mature CCA end. This step is carried out by RNase T, a member of the large DEDD family of exonucleases. We report the crystal structures of RNase T from Escherichia coli and Pseudomonas aeruginosa, which show that this enzyme adopts an opposing dimeric arrangement, with the catalytic DEDD residues from one monomer closely juxtaposed with a large basic patch on the other monomer. This arrangement suggests that RNase T has to be dimeric for substrate specificity, and agrees very well with prior site-directed mutagenesis studies. The dimeric architecture of RNase T is very similar to the arrangement seen in oligoribonuclease, another bacterial DEDD family exoribonuclease. The catalytic residues in these two enzymes are organized very similarly to the catalytic domain of the third DEDD family exoribonuclease in E. coli, RNase D, which is monomeric.     

A computational docking study for prediction of binding mode of diospyrin and derivatives: Inhibitors of human and leishmanial DNA topoisomerase-I

A computational approach was utilized to study the relative binding modes of diospyrin (bisnaphthoquinonoid) with the crystal structure of human DNA-TopoI and the recently reported Leishmania donavani DNA-TopoI. Additionally, the binding site interactions of amino derivatives of diospyrin with human TopoI were studied extensively. Based on the docking results, binding modes of diospyrin with the human and leishmanial TopoI catalytic core were predicted. The parallel use of two efficient and predictive docking programs, GOLD and Ligandfit, allowed mutual validation of the predicted binding poses. A reasonably good correlation coefficient between the calculated docking scores and the experimentally determined cytotoxicity helped in validating the docking method. Furthermore, a structure-based pharmacophore model was developed for L. donavani DNA-TopoI inhibition which helped in elucidating the topological and spatial requirements of the ligand-receptor interactions. This study provides an understanding of the structural basis of ligand binding to the topoisomerase receptor, which may be used for the structure-based design of potent and novel ligands for anticancer and antileishmanial therapy. To our knowledge, this is the first report of a binding mode exploration study for diospyrin and its derivatives as inhibitors of the leishmanial and human TopoI enzymes.     

Overexpression and purification of human calcitonin gene-related peptide-receptor component protein in Escherichia coli

Calcitonin gene-related peptide (CGRP) is a neuropeptide secreted by the central and peripheral nervous system nerves that has important physiological functions such as vasodilation, cardiotonic actions, metabolic and pro-inflammatory effects. The CGRP receptor is unique among G-protein coupled receptors in that a functional CGRP receptor consists of at least three proteins: calcitonin like receptor (CLR), receptor activity modifying protein (RAMP1) and receptor component protein (RCP). RCP is a required factor in CGRP-mediated signal transduction and it couples the CGRP receptor to the signal transduction pathway. Here, we describe methods to overexpress and purify RCP for structure-function studies. Human RCP was cloned and overexpressed with a poly-histidine tag and as a maltose binding protein (MBP) fusion in Escherichia coli using commercially available expression vectors. While His tagged RCP is prone to aggregation, solubility is improved when RCP is expressed as a MBP fusion. Expression and purification procedures for these constructs are described. Results from these studies will facilitate structural analysis of human RCP, and allow further understanding of RCP function.