S. M. Saqlan Naqvi

Germin and Germin-like Proteins: Evolution, Structure, and Function

Germin and germin-like proteins (GLPs) are encoded by a family of genes found in all plants. They are part of the cupin superfamily of biochemically diverse proteins, a superfamily that has a conserved tertiary structure, though with limited similarity in primary sequence. The subgroups of GLPs have different enzyme functions that include the two hydrogen peroxide–generating enzymes, oxalate oxidase (OxO) and superoxide dismutase. This review summarizes the sequence and structural details of GLPs and also discusses their evolutionary progression, particularly their amplification in gene number during the evolution of the land plants. In terms of function, the GLPs are known to be differentially expressed during specific periods of plant growth and development, a pattern of evolutionary subfunctionalization. They are also implicated in the response of plants to biotic (viruses, bacteria, mycorrhizae, fungi, insects, nematodes, and parasitic plants) and abiotic (salt, heat/cold, drought, nutrient, and metal) stress. Most detailed data come from studies of fungal pathogenesis in cereals. This involvement with the protection of plants from environmental stress of various types has led to numerous plant breeding studies that have found links between GLPs and QTLs for disease and stress resistance. In addition the OxO enzyme has considerable commercial significance, based principally on its use in the medical diagnosis of oxalate concentration in plasma and urine. Finally, this review provides information on the nutritional importance of these proteins in the human diet, as several members are known to be allergenic, a feature related to their thermal stability and evolutionary connection to the seed storage proteins, also members of the cupin superfamily.

Motility, acrosome integrity, membrane integrity and oocyte cleavage rate of sperm separated by swim-up or Percoll gradient method from frozen-thawed buffalo semen.

Frozen-thawed semen of five buffalo bulls was used to compare efficacy of swim-up and Percoll gradient methods for separating viable spermatozoa. Sperm separated by the two methods were also tested to differentiate buffalo bulls on the basis of in vitro fertilization (IVF) rates. Recovery of motile sperm (%), increase in membrane integrity (%) and acrosome integrity (%) were compared after two sperm separation methods in experiment I, and in vitro fertilization rate (cleavage rate and cleavage index) was compared in experiment II. Swim-up separated sperm showed a higher motility (P<0.05), while percent recovery of motile sperm was higher with Percoll separation (P<0.05). Membrane integrity (%) of sperm separated with swim-up was significantly higher (P<0.05) as compared to sperm separated with Percoll gradient. Swim-up separated sperm gave a higher cleavage rate and cleavage index (P<0.001). Sperm separated by swim-up showed significant difference among the bulls in cleavage rate and cleavage index (P<0.05), while the Percoll gradient method did not. It has been concluded that separation of sperm from frozen-thawed buffalo semen by swim-up method can be more expedient for IVF in buffalo.

An overview on the small heat shock proteins

In the last 25 years, a huge amount of literature has been accumulated describing the cell’s response to different kinds of environmental stress conditions, such as high temperatures, altered pH, exposure of the cell to toxins, starvation, oxygen, and water deprivation, among others. Heat shock proteins (HSPs) are one of the main expressed products of the cell in response to stresses. HSPs can be classified into six structurally conserved classes according to their molecular weight namely, HSP100, HSP90, HSP70, HSP60, small heat shock proteins (sHSPs) and ubiquitin (8.5 kDa). In eukaryotes, different heat shock genes are expressed uncoordinatedly, whereas in prokaryote, heat shock genes form a regulon and appear simultaneously. sHSPs are associated with nuclei, cytoskeleton and membranes. They bind partially to denatured proteins, preventing irreversible protein aggregation during stress. In animals, only one sHSP gene has been located in yeast cells, ten in mammalian, two in birds and four genes have been found in Drosophila . However, in plants more than 20 sHSPs have been reported and they can be divided into 6 classes, of which, 3 classes (CI, CII and CIII) are in the cytosole or in the nucleus and the other three (CIV, CV and CVI) in the plastids, endoplasmic reticulum and mitochondria. Mitochondrial and chloroplast sHSPs protect electron transport chain. During development in animals, sHSP genes are normally regulated at late neurula and early tailbud stage and in plants during pollen development, seed maturation, seed imbibition and germination. Transcriptional regulation of sHSPs depends on particular activation of heat shock factors (HSF) which recognize the highly conserved heat-shock elements (HSEs). After the heat stress has been released, the sHSPs are quite stable, suggesting that sHSPs may be important for recovery as well.

Cloning and sequence analysis of germin-like protein gene 2 promoter from Oryza sativa L. ssp. indica

Germin and germin-like proteins (GLPs) are water soluble extracellular proteins reportedly expressed in response to some environmental and developmental signals. Some enzymatic activities have also been associated with germin/GLPs. However, their role in overall metabolism has not been fully understood. Significant insight into their function may also be gained by analysis of their promoter. During this study, about 1107 bp 5′region of OsRGLP2 gene was amplified, cloned and sequenced. The sequence analysis by BLAST showed that this promoter sequence has five common regions (CR1–CR5) of different sizes, which are repeated at 3–6 other locations in 30 kb region in which this gene driven by its promoter is located. Interestingly, all the genes driven by promoter harboring these common regions are GLPs/putative germins. Analysis of these common regions located on OsRGLP2 indicated presence of many elements including those for light responsiveness, dehydration and dark induced senescence, stresses (pathogen and salt), plant growth regulators, pollen specific expression and elements related to seed storage proteins. Analysis of the 30 kb germin/GLP clustered region by GenScan detected each gene to have a putative 40 bp promoter which contains TATA box and Dof factor which turned out to be a part of CR2.

Structural basis of nucleic acid binding by Nicotiana tabacum glycine-rich RNA-binding protein: implications for its RNA chaperone function

Glycine-rich RNA-binding proteins (GR-RBPs) are involved in cold shock response of plants as RNA chaperones facilitating mRNA transport, splicing and translation. GR-RBPs are bipartite proteins containing a RNA recognition motif (RRM) followed by a glycine-rich region. Here, we studied the structural basis of nucleic acid binding of full-length Nicotiana tabacum GR-RBP1. NMR studies of NtGR-RBP1 show that the glycine-rich domain, while intrinsically disordered, is responsible for mediating self-association by transient interactions with its RRM domain (NtRRM). Both NtGR-RBP1 and NtRRM bind specifically and with low micromolar affinity to RNA and single-stranded DNA. The solution structure of NtRRM shows that it is a canonical RRM domain. A HADDOCK model of the NtRRM–RNA complex, based on NMR chemical shift and NOE data, shows that nucleic acid binding results from a combination of stacking and electrostatic interactions with conserved RRM residues. Finally, DNA melting experiments demonstrate that NtGR-RBP1 is more efficient in melting CTG containing nucleic acids than isolated NtRRM. Together, our study supports the model that self-association of GR-RBPs by the glycine-rich region results in cooperative unfolding of non-native substrate structures, thereby enhancing its chaperone function.

Multiple pulses improve electroporation efficiency in Agrobacterium tumefaciens

Electroporation entails brief, high intensity pulse to create transient pores in the cell membrane to facilitate the entry of exogenous macromolecules, which may otherwise be excluded. Removal of the external field leads to the resealing of the membrane electropores permitting the survival of the electrically stimulated recipient cells. Using this technique foreign deoxyribonucleic acid (DNA) has been successfully introduced into many cell types both from prokaryotes and eukaryotes. Increase in pulse voltage and length beyond a critical limit has been reported to decrease transformation efficiency, hence in this study we have investigated another strategy i.e. increase in the number of pulses at constant high voltage and pulse duration. Commonly used Agrobacterium strains LBA4404 and EHA101 and binary vector pCAMBIA1301 were used. Transformants were selected on a combination of hygromycin and kanamycin, and confirmed by polymerase chain reaction (PCR) and restriction analysis. Increase in the number of pulses was found to show a significant and linear increase in transformation efficiency.

Comparative Assessment of Synthetic-derived and Conventional Bread Wheat Advanced Lines Under Osmotic Stress and Implications for Molecular Analysis

Drought is one of the most important environmental factors limiting wheat yield in many parts of the world. Progress in breeding to improve drought tolerance has been limited by its high sensitivity to environmental factors, low heritability, and the complexity and size of wheat genome. Two genetically diverse sets of wheat genotypes were evaluated to identify genetic resources maintaining physiological and metabolic stability under osmotic stress. Data on 13 different morphological and physiological traits under control and osmotic stress clearly depicted the superiority of wheat lines derived from synthetic hexaploid wheats (SHWs) as compared to conventional bread wheats. Accordingly, all lines were genotyped with simple sequence repeat (SSR) markers to assess the diversity and identify the marker–trait associations (MTAs). Structure analysis partitioned the germplasm into two sub-populations (K = 2) based on ΔK and LnP(D) values. Association mapping was performed using Q + K matrix as covariates by applying mixed linear model (MLM). In total, 39 MTAs over 20 SSR loci were detected by the strict MLM model, which were reduced to 12 MTAs over 6 SSR loci after strict Bonferroni adjustments. MTAs detected under osmotic stress conditions indicated the effectiveness of association mapping to identify loci for different attributes under low-moisture conditions. Conclusively, this study has demonstrated that synthetic-derived wheats harbor valuable alleles that can enrich the genetic base of cultivated wheat and improve its adaptation under water stress conditions. The MTAs detected may have the candidate genes responsible for drought adaptation, thus providing a unique resource which can be manipulated for developing drought-tolerant cultivars.

Phylogenetic relationship of TJ1 isolate of Banana bunchy top virus from Pakistan by DNA-R sequence analysis

The disease caused by the Banana bunchy top virus (BBTV) is a major problem in banana crops. It spreads either through vegetative propagation of the plant and (or) by means of a vector, Pentalonia nigronervosa. Planting material for banana cultivation was introduced to Sindh (a major banana-growing province of Pakistan) from many countries where BBTV was present. A recent BBTV epidemic in the Sindh region resulted in considerable damage to the banana crop. To determine the probable origin of this epidemic, BBTV was isolated from bananas in Sindh, and the DNA-R of the resulting TJ1 isolate was cloned, sequenced, and subjected to phylogenetic analysis. Analysis with the complete sequence of the DNA-R showed that the TJ1 isolate is a member of the South-Pacific group. Analysis with the coding or the conserved regions of the DNA-R supported the same grouping. The TJ1 isolate shows the highest homology with the Egyptian isolate within this group, followed by the Australian and Indian isolates. All these isolates seem to have evolved from a common ancestor.

Sperm sexing in Nili-Ravi buffalo through modified swim up: Validation using SYBR® green real-time PCR

Sperm sexing through flow-sorting technology is relatively expensive, requires considerable technical support and is actually not practicable in many developing countries. The aim of this study was to investigate the feasibility of producing enriched pools of X or Y chromosome-bearing sperm by a modified swim-up method. For this purpose semen was collected from five mature Nili-Ravi buffalo bulls for a period of six weeks. The qualifying ejaculates were divided into two aliquots for further processing through modified swim-up or control (untreated). After processing, semen was cryopreserved in tris citric acid extender using standard techniques. Semen quality was assessed at pre dilution, post dilution and post thawing. Validation of technique was done by using SYBR® green real time PCR using two sets of primers, PLP and SRY for X and Y chromosome of buffalo genes, respectively. Sperm recovery rates, pre freeze and post thaw sperm quality were found significantly higher in X chromosome bearing sperm fraction than Y chromosome bearing fraction and control. Mean fold relative expression of X bearing sperm was significantly higher (4-5 fold) in X chromosome bearing fraction of supernatant than Y chromosome bearing fraction (0.06 fold), similarly mean fold relative expression of Y chromosome bearing sperm was significantly higher in Y chromosome bearing fraction (4 fold) of supernatant than X chromosome bearing fraction (0.15 fold) compared to control (1.00). In conclusion, a modified swim up method proved to be an effective method for Nili-Ravi buffalo sperm sexing as validated by real time PCR.

The Incidence of Alternaria Species Associated with Infected Sesamum indicum L. Seeds from Fields of the Punjab, Pakistan

Sesame (Sesamum indicum) is an important oil seed crop of Asia. Yields can be negatively impacted by various factors, including disease, particularly those caused by fungi which create problems in both production and storage. Foliar diseases of sesame such as Alternaria leaf blight may cause significant yield losses, with reductions in plant health and seed quality. The work reported here determined the incidence of Alternaria species infecting sesame seeds grown in the Punjab, Pakistan. A total of 428 Alternaria isolates were obtained from 105 seed samples and grouped into 36 distinct taxonomic groups based on growth pattern and morphological characters. Isolation frequency and relative density of surface sterilized and non-surface sterilized seeds showed that three isolates (A13, A47 and A215) were the most common morphological groups present. These isolates were further identified using sequencing of the Internal Transcribed Spacer (ITS) region of ribosomal DNA (rDNA) and the Alternaria major allergen gene (Alt a 1). Whilst ITS of rDNA did not resolve the isolates into Alternaria species, the Alt a 1 sequences exhibited > 99% homology with Alternaria alternata (KP123850.1) in GenBank accessions. The pathogenicity and virulence of these isolates of Alternaria alternata was confirmed in inoculations of sesame plants resulting in typical symptoms of leaf blight disease. This work confirms the identity of a major source of sesame leaf blight in Pakistan which will aid in formulating effective disease management strategies.