Zymographic Method for Distinguishing Different Classes of Superoxide Dismutases in Plants.

In plants, especially in chloroplasts, superoxide radical is generated when an electron is transferred to dimolecular O2 due to decreased activity of Photosystem I. The superoxide (O2-) radical accumulation is more rampant in plants exposed to abiotic stresses due to oxidation of photosystem components. Excessive superoxide radical accumulation will lead to oxidative damage to the cellular macromolecules. The ubiquitous superoxide dismutases (SODs) represent critical enzymatic antioxidant system present in cells, which can catalyze the disproportion of superoxide (O2-) radical rapidly into hydrogen peroxide (H2O2) and molecular oxygen. Depending on the metal cofactor present, the plant SODs are classified into Cu/ZnSOD, MnSOD, and FeSOD. The activity of SODs can be quantified zymographically. Additionally, using this method, different classes of SODs can be distinguished by using H2O2, KCN, and NaN3.

Characterization of drought- and heat-responsive microRNAs in switchgrass.

Recent investigations revealed that microRNAs (miRNAs) play crucial roles in plant acclimation to stress conditions. Switchgrass, one of the important biofuel crop species can withstand hot and dry climates but the molecular basis of stress tolerance is relatively unknown. To identify miRNAs that are important for tolerating drought or heat, small RNAs were profiled in leaves of adult plants exposed to drought or heat. Sequence analysis enabled the identification of 29 conserved and 62 novel miRNA families. Notably, the abundances of several conserved and novel miRNAs were dramatically altered following drought or heat. Using at least one fold (log2) change as cut off, we observed that 13 conserved miRNA families were differentially regulated by both stresses, and, five and four families were specifically regulated by drought and heat, respectively. Similarly, using a more stringent cut off of two fold (log2) regulation, we found 5 and 16 novel miRNA families were upregulated but 6 and 7 families were downregulated under drought and heat, respectively. The stress-altered expression of a subset of miRNAs and their targets was confirmed using quantitative PCR. Overall, the switchgrass plants exposed to drought or heat revealed similarities as well as differences with respect to miRNA regulation, which could be important for enduring different stress conditions.

Small RNA profiles in soybean primary root tips under water deficit.

BACKGROUND: Soybean (Glycine max) production is significantly hampered by frequent droughts in many regions of the world including the United States. Identifying microRNA (miRNA)-controlled posttranscriptional gene regulation under drought will enhance our understanding of molecular basis of drought tolerance in this important cash crop. Indeed, miRNA profiles in soybean exposed to drought were studied but not from the primary root tips, which is not only a main zone of water uptake but also critical for water stress sensing and signaling. METHODS: Here we report miRNA profiles specifically from well-watered and water-stressed primary root tips (0 to 8 mm from the root apex) of soybean. Small RNA sequencing confirmed the expression of vastly diverse miRNA (303 individual miRNAs) population, and, importantly several conserved miRNAs were abundantly expressed in primary root tips. RESULTS: Notably, 12 highly conserved miRNA families were differentially regulated in response to water-deficit; six were upregulated while six others were downregulated at least by one fold (log2) change. Differentially regulated soybean miRNAs are targeting genes include auxin response factors, Cu/Zn Superoxide dismutases, laccases and plantacyanin and several others. CONCLUSIONS: These results highlighted the importance of miRNAs in primary root tips both under control and water-deficit conditions; under control conditions, miRNAs could be important for cell division, cell elongation and maintenance of the root apical meristem activity including quiescent centre whereas under water stress differentially regulated miRNAs could decrease auxin signaling and oxidative stress as well as other metabolic processes that save energy and water.

High throughput sequencing of small RNA component of leaves and inflorescence revealed conserved and novel miRNAs as well as phasiRNA loci in chickpea.

Among legumes, chickpea (Cicer arietinum L.) is the second most important crop after soybean. MicroRNAs (miRNAs) play important roles by regulating target gene expression important for plant development and tolerance to stress conditions. Additionally, recently discovered phased siRNAs (phasiRNAs), a new class of small RNAs, are abundantly produced in legumes. Nevertheless, little is known about these regulatory molecules in chickpea. The small RNA population was sequenced from leaves and flowers of chickpea to identify conserved and novel miRNAs as well as phasiRNAs/phasiRNA loci. Bioinformatics analysis revealed 157 miRNA loci for the 96 highly conserved and known miRNA homologs belonging to 38 miRNA families in chickpea. Furthermore, 20 novel miRNAs belonging to 17 miRNA families were identified. Sequence analysis revealed approximately 60 phasiRNA loci. Potential target genes likely to be regulated by these miRNAs were predicted and some were confirmed by modified 5' RACE assay. Predicted targets are mostly transcription factors that might be important for developmental processes, and others include superoxide dismutases, plantacyanin, laccases and F-box proteins that could participate in stress responses and protein degradation. Overall, this study provides an inventory of miRNA-target gene interactions for chickpea, useful for the comparative analysis of small RNAs among legumes.

Characterization of a chemostable serine alkaline protease from Periplaneta americana.

BACKGROUND: Proteases are important enzymes involved in numerous essential physiological processes and hold a strong potential for industrial applications. The proteolytic activity of insects' gut is endowed by many isoforms with diverse properties and specificities. Thus, insect proteases can act as a tool in industrial processes. RESULTS: In the present study, purification and properties of a serine alkaline protease from Periplaneta americana and its potential application as an additive in various bio-formulations are reported. The enzyme was purified near to homogeneity by using acetone precipitation and Sephadex G-100 gel filtration chromatography. Enzyme activity was increased up to 4.2 fold after gel filtration chromatography. The purified enzyme appeared as single protein-band with a molecular mass of ~ 27.8 kDa in SDS-PAGE. The optimum pH and temperature for the proteolytic activity for purified protein were found around pH 8.0 and 60°C respectively. Complete inhibition of the purified enzyme by phenylmethylsulfonyl fluoride confirmed that the protease was of serine-type. The purified enzyme revealed high stability and compatibility towards detergents, oxidizing, reducing, and bleaching agents. In addition, enzyme also showed stability towards organic solvents and commercial detergents. CONCLUSION: Several important properties of a serine protease from P. Americana were revealed. Moreover, insects can serve as excellent and alternative source of industrially important proteases with unique properties, which can be utilized as additives in detergents, stain removers and other bio-formulations. Properties of the P. americana protease accounted in the present investigation can be exploited further in various industrial processes. As an industrial prospective, identification of enzymes with varying essential properties from different insect species might be good approach and bioresource.

Alterations in the Helicoverpa armigera midgut digestive physiology after ingestion of pigeon pea inducible leucine aminopeptidase.

Jasmonate inducible plant leucine aminopeptidase (LAP) is proposed to serve as direct defense in the insect midgut. However, exact functions of inducible plant LAPs in the insect midgut remain to be estimated. In the present investigation, we report the direct defensive role of pigeon pea inducible LAP in the midgut of Helicoverpa armigera (Lepidoptera: Noctuidae) and responses of midgut soluble aminopeptidases and serine proteinases upon LAP ingestion. Larval growth and survival was significantly reduced on the diets supplemented with pigeon pea LAP. Aminopeptidase activities in larvae remain unaltered in presence or absence of inducible LAP in the diet. On the contrary, serine proteinase activities were significantly decreased in the larvae reared on pigeon pea LAP containing diet as compared to larvae fed on diet without LAP. Our data suggest that pigeon pea inducible LAP is responsible for the degradation of midgut serine proteinases upon ingestion. Reduction in the aminopeptidase activity with LpNA in the H. armigera larvae was compensated with an induction of aminopeptidase activity with ApNA. Our findings could be helpful to further dissect the roles of plant inducible LAPs in the direct plant defense against herbivory.

Wound and methyl jasmonate induced pigeon pea defensive proteinase inhibitor has potency to inhibit insect digestive proteinases.

Wounding of plants by chewing insects or other damage induces the synthesis of defensive proteinase inhibitors (PI) in both wounded and distal unwounded leaves. In the present paper we report the characterization of inducible defensive PI from pigeon pea (Cajanus cajan) and its in vitro interaction with Helicoverpa armigera gut proteinases (HGP). We found that PI activity was induced in local as well as systemic leaves of pigeon pea by the wounding and methyl jasmonate (MeJA) application. Consistent induction of PI was observed in two wild cultivars of pigeon pea at various growth stages. The estimated molecular weight of inducible PI was ~16.5 kDa. Electrophoretic analysis and enzyme assays revealed that the induced PI significantly inhibited total gut proteinase as well as trypsin-like activity from the midgut of H. armigera. The induced PI was found to be inhibitor of trypsin as well as chymotrypsin. Study could be important to know the further roles of defensive PIs.

Changes and induction of aminopeptidase activities in response to pathogen infection during germination of pigeonpea (Cajanas cajan) seeds.

Aminopeptidases play important role in the mobilization of storage proteins at the cotyledon during seed germination. It is often referred as inducible component of defense against herbivore attack. However the role of aminopeptidase in response to pathogen attack in germinating seeds is remained to be unknown. An attempt was made to analyze change in the aminopeptidase (EC 3.4.11.1) activity during germination of pigeonpea (Cajanus cajan L.) seeds by infecting the seeds with fungi. Two aminopeptidase activity bands (AP1 and AP2) were detected in control as well as infected pigeonpea seeds. During latter stages of germination in control seeds, AP1 activity was replaced by AP2 activity. However AP1 activity was significantly induced in germinating seeds infected with Fusarium oxysporum f.sp. ciceri and Aspergillus niger var. niger. The estimated molecular weights of AP1 and AP2 were ∼97 and 42.8kDa respectively. The induced enzyme was purified up to 30 fold by gel filtration chromatography. The purified enzyme was preferentially cleaved leucine p-nitroanilide than alanine p-nitroanilide. The enzyme was strongly inhibited by bestatin and 1,10-phenanthroline. Almost 50% of enzyme activity was inhibited by ethylene diamine tetra acetate. The purified enzyme showed broad pH optima ranging from pH 6.0 to 9.0 and optimum at pH 8.5. The induction of aminopeptidase activity during pigeonpea seed germination and in response to pathogen attack indicates significant involvement of these enzymes in primary as well as secondary metabolism of the seeds. These findings could be helpful to further dissect defensive role of aminopeptidases in seed germination which is an important event in plant's life.

Biochemical characterisation of α-amylase inhibitors from Achyranthes aspera and their interactions with digestive amylases of coleopteran and lepidopteran insects.

BACKGROUND: Starchy seeds are an important food and a source of dietary ingredients in many countries. However, they suffer from extensive predation by bruchids (weevils) and other pests. α-Amylase inhibitors are attractive candidates for the control of seed weevils, as these insects are highly dependent on starch as an energy source. RESULTS: A proteinaceous α-amylase inhibitor from the seeds of Achyranthes aspera was identified, purified and characterised. In electrophoretic analysis, two prominent amylase inhibitor activity bands (AI1 and AI2) were detected. The inhibitor was purified 9.99-fold with 1206.95 total amylase inhibitor units mg⁻¹ protein. The molecular weight of the purified inhibitor was around 6 kDa. The isolated α-amylase inhibitor was found to be resistant to heat and proteolysis. Feeding analysis of Callosobruchus maculatus larvae on a diet containing seed powder of A. aspera revealed that survival of the larvae was severely affected, with the highest mortality rate occurring on the fifth day of feeding. The isolated inhibitor inhibited the majority of amylase isoforms of C. maculatus, Tribolium confusum and Helicoverpa armigera in electrophoretic analysis and solution assays. CONCLUSION: The information obtained in the present investigation could be useful for a genetic engineering approach that would make seeds resistant to storage pest infestations.

Induction of leucine aminopeptidase (LAP) like activity with wounding and methyl jasmonate in pigeonpea (Cajanas cajan) suggests the role of these enzymes in plant defense in leguminosae.

Aminopeptidases are ubiquitous in nature and their activities have been identified in several plant species. Leucine aminopeptidases (LAPs) are predominantly studied in solanaceous plants and are induced in response to wounding, herbivory and methyl jasmonate (MeJA). The functions of plant aminopeptidases are still under discussion and it is likely that the different classes play various roles. In the present study we report the local and systemic induction of LAP-like activity upon mechanical wounding and MeJA treatment. Two proteins with LAP-like activity were detected in pigeonpea leaves. They were designated as AP1 and AP2. AP1 activity was significantly induced upon wounding and application of MeJA. The estimated molecular masses of AP1 and AP2 were ∼ 60 and 41 kDa respectively in SDS-PAGE. The pH optimum for LAP-like activity in control leaf extracts was found to be neutral (pH 7.0) however the enzymes showed highest activity at alkaline pH (pH 9.0) in the leaf extracts of treated plants. The temperature optimum for LAP-like activity was around 40-50 °C. The enzymes were strongly inhibited by 1, 10 phenanthroline and bestatin. Heavy metal ions and EDTA inhibited LAP-like activities, whereas Mn(+2) and Mg(+2) activated the enzyme activities. Beside LpNA (33.5 U/mg/min) pigeonpea LAP-like enzymes also cleaved ApNA (15 U/mg/min) but were unable to cleave VpNA. Total proteolytic activity was also observed to be induced in treated plants. LAP-like activity was increased upto 19.5 fold after gel filtration chromatography. Results suggest that these enzymes may have functional defensive role in pigeonpea.

Partial purification and characterization of Helicoverpa armigera (Lepidoptera: Noctuidae) active aminopeptidase secreted in midgut.

We report the partial purification to apparent homogeneity of a soluble aminopeptidase (EC 3.4.11.1) from midgut of Helicoverpa armigera larvae, which preferentially degraded Leucine p-nitroanilide (LpNA). After midgut isolation, extraction and precipitation of soluble proteins with acetone, proteins were purified in two consecutive steps including gel filtration and ion-exchange chromatographies. Aminopeptidase activity was increased 8.95 fold after gel filtration chromatography. The purified enzyme appeared as single band with a molecular mass of approximately 112 kDa in SDS-PAGE, with a pH optimum of 7.0. Zymogram analysis revealed two enzymatically active proteinases using LpNA as substrate. The optimal temperature of aminopeptidase activity was 50-60 degrees C. The enzyme was characterized as metalloprotease as it was strongly inhibited by 1,10 phenanthroline. Strong inhibition was also being observed using the specific aminopeptidase inhibitor bestatin. Heavy metal ions, EDTA and cysteine strongly inhibited the enzyme, while Ca(+2), Mn(+2) and Mg(+2) somewhat stimulated aminopeptidase activity. Besides LpNA, the purified aminopeptidase also cleaved with decreasing activity ApNA, VpNA and BApNA. Study could be helpful to understand the mechanism of action of N-terminal degrading enzymes and also important is to further study the differential interaction of Bacillus thuringiensis cry insecticidal toxin with midgut receptor of insects.

Unraveling biochemical properties of cockroach (Periplaneta americana) proteinases with a gel X-ray film contact print method.

Eleven proteinase activity bands were detected in American cockroach (Periplaneta americana) gut. These were partially purified and characterized using a gel X-ray film contact print method. Cockroach gut proteinases (CGPs) show activity over a broad range of pH with maximum activity between pH 6 and 10, and optimal activity at 50-70 degrees C. CGPs were partially purified by preparative gel electrophoresis and analyzed using synthetic substrates and inhibitors. Four of the proteases exhibited chymotrypsin-like (C1 to C4) activity and seven trypsin-like (T1 to T7) activity. Accuracy of the gel X-ray film contact print method is confirmed by including bovine chymotrypsin in CGP analysis. Inhibition of CGPs with different plant proteinaceous proteinase inhibitors allowed identification of potential CGP inhibitors. Our results imply that presence of several CGP activity bands, and their stability and activity over a broad pH and temperature range might contribute to adaptation of P. americana to extreme environmental conditions and the polyphagous nature of the species.

Differential inhibition of Helicoverpa armigera gut proteinases by proteinase inhibitors of pigeonpea (Cajanus cajan) and its wild relatives.

The seeds of 36 pigeonpea [Cajanus cajan (L) Millsp.] cultivars, resistant and susceptible to pests and pathogens and 17 of its wild relatives were analysed for inhibitors of trypsin, chymotrypsin, and insect gut proteinases to identify potential inhibitors of insect (Helicoverpa armigera) gut enzymes. Proteinase inhibitors (PIs) of pigeonpea cultivars showed total inhibition of trypsin and chymotrypsin, and moderate inhibition potential towards H. armigera proteinases (HGP). PIs of wild relatives exhibited stronger inhibition of HGP, which was up to 87% by Rhynchosia PIs. Electrophoretic detection of HGPI proteins and inhibition of HGP isoforms by few pigeonpea wild relative PIs supported our enzyme inhibitor assay results. Present results indicate that PIs exhibit wide range of genetic diversity in the wild relatives of pigeonpea whereas pigeonpea cultivars (resistant as well as susceptible to pests and pathogens) are homogeneous. The potent HGPIs identified in this study need further exploration for their use in strengthening pigeonpea defence against H. armigera.