Prabhjeet Singh

DNA isolation from dry and fresh samples of polysaccharide-rich plants

DNA extraction is difficult in a variety of plants because of the presence of metabolites that interfere with DNA isolation procedures and downstream applications such as DNA restriction, amplification, and cloning. The chemotypic heterogeneity among species may not permit optimal DNA yield with a single protocol; thus, even closely related species may require different isolating protocols. Here we describe a modified procedure based on the hexadecyltrimethylammonium bromide (CTAB) method to isolate DNA from tissues containing high levels of polysaccharides. The procedure is applicable to both dry and fresh tissues and was tested on chickpea seeds, soybean, and wheat leaves. This method solved the problems of DNA degradation, contamination, and low yield due to binding and/or coprecipitation with starches and polysaccharides. The isolated DNA proved amenable to PCR amplification and restriction digestion.

Changes in germination, growth and soluble sugar contents of Sorghum bicolor (L.) Moench seeds under various abiotic stresses

The effect of various abiotic stresses on germination rate, growth and soluble sugar content in Sorghum bicolor (L.) Moench cv. CSH 6 seed embryos and endosperm during early germination was investigated. Under stress conditions germination, water potential and tissue water content decreased markedly. Subsequently, this reduction resulted in marked decreases in fresh weight both in embryos and endosperm. Conversely, a substantial increase in dry weight was observed. Furthermore, a considerable increase in the sugar contents in both embryo and endosperm was detected. The fructose level was always higher than glucose and sucrose in response to various stresses. However, as compared to the control the level of glucose and sucrose was higher in embryos and endosperm after stress treatments. Based upon these results a possible physiological role of sugars in the germination of sorghum seeds is discussed.

Effect of media supplements and culture conditions on inulinase production by an actinomycete strain

Streptomyces sp. GNDU 1 produced high levels of extra-cellular inulinase (0.552 IU/ml) after 24 h at pH 7.5, temperature 46 degrees C in the presence of 1% inulin. The optimum temperature and pH for enzyme activity were 60 degrees C and 5.5 respectively. Yeast extract as a nitrogen source was found to be most suitable one for inulinase production whereas ammonium ion was inhibitory to the enzymatic production. All these conditions make Streptomyces sp. GNDU 1, a potential candidate for industrial enzymatic production of fructose from inulin.

Cyclophilins: Proteins in search of function

Cyclophilins constitute a subgroup of large family of proteins called immunophilins, which also include FKBPs and Parvulins. They are remarkably conserved in all genera, highlighting their pivotal role in important cellular processes. Most cyclophilins display PPIase enzymatic activity, multiplicity, diverse cellular locations and active role in protein folding which render them to be included in the class of diverse set of proteins called molecular chaperones. Due to their distinct PPIase function, besides protein disulfide isomerases and protein foldases, cyclophilins have been deemed necessary for in vivo chaperoning activity. Unlike other cellular chaperones, these proteins are specific in their respective targets. Not all cyclophilin proteins possess PPIase activity, indicating a loss of their PPIase activity during the course of evolution and gain of function independent of their PPIase activity. The PPIase function of cyclophilins is also compensated by their functional homologs, like FKBPs. Multiple cyclophilin members in plants like Arabidopsis and rice have been reported to be associated with diverse functions and regulatory pathways through their foldase, scaffolding, chaperoning or other unknown activities. Although many functions of plant cyclophilins were reported or suggested, the physiological relevance and molecular basis of stress-responsive expression of plant cyclophilins is still largely unknown. However, their wide distribution and ubiquitous nature signifies their fundamental importance in plant survival. Several of these members have also been directly linked to multiple stresses. This review attempts to deal with plant cyclophilins with respect to their role in stress response.

A quick method to isolate RNA from wheat and other carbohydrate-rich seeds

No reports on isolating RNA from carbohydrate-rich wheat seeds have been published. Because of the presence of carbohydrates, published protocols yield small amounts of poor quality RNA. Extracting seeds in a buffer (pH 9, 150 mM NaCl, 1% sarcosyl) ensured maximum RNA solubility and the removal of most interfering substances. Extracted RNA was purified using a guanidine hydrochloride-based buffer system. This protocol yields up to 148 μg of RNA from 100 mg of tissue in 3.5 h. An A260/A280 ratio of 1.85 indicates RNA purity. Isolated RNA was amenable to downstream applications such as differential display. The developed method was extended to other carbohydrate-rich seeds, such as barley and maize, with success.

RNA isolation from plant tissues rich in polysaccharides

Extraction of high-quality RNA is necessary formakingcDNAlibraries,isolatinggenesbyRT-PCR,orinvestigatinggeneexpressionprofiles.SeveralmethodsarecommonlyusedforisolationoftotalRNA[1–3]andare being developed because plants contain highamountsofmanydifferentsubstances;therefore,justonenucleicacidisolationmethodsuitableforallplantscanneverexist[4].Evenplantspeciesbelongingtothesamegenusorrelatedgeneracanexhibitenormousvariabilityinthecomplexityofpathwaysofdispensablefunctions.Thus,thebiochemicalcompositionsinplanttissuesofdifferentspeciesareexpectedtovaryconsid-erably.ThechemotypicheterogeneityamongspeciesmaynotallowoptimalRNAyieldfromoneisolationprotocoland,perhaps,evencloselyrelatedspeciesmayrequiredifferentisolationprotocols[5].Asouraimhasbeentostudydrought-induceddifferentialexpressionofgenesduringearlyandlateseeddevelopmentalstagesinsorghum,wemayneedaprotocolthatnotonlycangivethesamequalityandquantityofRNAateachstagebutalsoishighyieldinginordernottomisstherarelyex-pressedgenes.PreviousstandardplantRNAisolationproceduresfailedtoworkwhenappliedtotissuesrichinsecondaryproducts[3,6–8].Inthisreport,aproceduredevelopedbyLogemannetal.[3]wasmodifiedandappliedtosorghumseeds,whichareknowntocontainhighlevelsofpolysaccharides[9].Theextractionmethoddescribedinthisstudyissimple,notrequiringultra-centrifugationoradditionalprecipitationsteps,andal-lowsRNAextractionfromplantspeciesinwhichotherprocedureshavepreviouslybeenunsuccessful.Further,thequalityoftheisolatedRNAwasconsistentlyhighasindicatedbyspectrophotometricreadingsanditssepa-rationondenaturingagarosegels.TheyieldandqualityweresuitableforRT-PCRandNorthernblothybrid-ization.Seeds of sorghum (Sorghum bicolor cv. CSH-6)werepurchasedfromtheNationalSeedCorp.,(Pusa,NewDelhi,India)andseedsofchickpea(Cicerarieti-num),andsoybean(Glycinemax)werepurchasedfromPunjabAgriculturalUniversity(Ludhiana,India).Theseedsweresurfacesterilizedwith1%(w/v)mercuricchloride and 70% ethanol followed by rinsing withdeionizedwater.Seedswereimbibedfor6hindouble-distilledwaterat37 Candusedforfurtherstudies.Leavesofsoybeanandchickpeawerecollectedfrom6-day-oldgerminatedseedlingsgrowninaseedgermin-ater.Tissueswerestoredinliquidnitrogenforfurtheranalysis.Solutionsandreagentsusedwereasfollows:ho-mogenization solution (Solution I), NaCl (5M),Sarkosyl(2%);guanidinehydrochloridebuffer(pH7.0),8Mguanidinehydrochloride,20mMEDTA,20mMMes,

Purification and characterization of an exoinulinase from Aspergillus fumigatus.

An extracellular exoinulinase was purified from the crude extract of Aspergillus fumigatus by ammonium sulfate precipitation, followed by successive chromatographies on DEAE-Sephacel, Sephacryl S-200, concanavalin A-linked amino-activated silica, and Sepharose 6B columns. The enzyme was purified 25-fold, and the specific activity of the purified enzyme was 171 IU/mg of protein. Gel filtration chromatography revealed a molecular weight of about 200 kDa, and native polyacrylamide gel electrophoresis (PAGE) showed an electrophoretic mobility corresponding to a molecular weight of about 176.5 kDa. Sodium dodecyl sulfate-PAGE analysis revealed three closely moving bands of about 66, 62.7, and 59.4 kDa, thus indicating the heterotrimeric nature of this enzyme. The purified enzyme appeared as a single band on isoelectric focusing, with a pI of about 8.8. The enzyme activity was maximum at pH 5.5 and was stable over a pH range of 4.0–9.5, and the optimum temperature for enzyme activity was 60°C. The purified enzyme retained 35.9 and 25.8% activities after 4 h at 50 and 55°C, respectively. The inulin hydrolysis activity was completely abolished with 1 mM Hg++, whereas EDTA inhibited about 63% activity. As compared to sucrose, stachyose, and raffinose, the purified enzyme had lower Km (0.25 mM) and higher Vmax (333.3 IU/mg) values for inulin.

Effect of Water Stress on Expression of a 20 kD Cyclophilin-like Protein in Drought Susceptible and Tolerant Cultivars of Sorghum

Immunodetection studies revealed the presence of a 20 kD cyclophilin-like protein (designated as SorgCyp20) in leaves and seeds of sorghum (Sorghum bicolor L Moench). The expression of SorgCyp20 was temporally regulated in the leaves and after attaining maximum levels at either 60 or 70 days after sowing it declined after flowering. The effect of drought stress on SorgCyp20 levels in the leaves and seeds of sorghum was stage and cultivar dependent. The drought stress-induced enhancement in SorgCyp20 levels was many times higher in the leaves (3-fold increase at 30 days after sowing) and seeds (2.5-fold increase at 9 days post anthesis) of drought tolerant cv ICSV-272 than in the drought susceptible cv SPRU-94008B. The intercultivar differences in drought stress-induced changes in SorgCyp20 expression were not related to the difference in water potential thus suggesting differential regulation of SorgCyp20 in response to stress in the two sorghum cultivars.

Abiotic stress responses in plants: roles of calmodulin-regulated proteins

Intracellular changes in calcium ions (Ca2+) in response to different biotic and abiotic stimuli are detected by various sensor proteins in the plant cell. Calmodulin (CaM) is one of the most extensively studied Ca2+-sensing proteins and has been shown to be involved in transduction of Ca2+ signals. After interacting with Ca2+, CaM undergoes conformational change and influences the activities of a diverse range of CaM-binding proteins. A number of CaM-binding proteins have also been implicated in stress responses in plants, highlighting the central role played by CaM in adaptation to adverse environmental conditions. Stress adaptation in plants is a highly complex and multigenic response. Identification and characterization of CaM-modulated proteins in relation to different abiotic stresses could, therefore, prove to be essential for a deeper understanding of the molecular mechanisms involved in abiotic stress tolerance in plants. Various studies have revealed involvement of CaM in regulation of metal ions uptake, generation of reactive oxygen species and modulation of transcription factors such as CAMTA3, GTL1, and WRKY39. Activities of several kinases and phosphatases have also been shown to be modulated by CaM, thus providing further versatility to stress-associated signal transduction pathways. The results obtained from contemporary studies are consistent with the proposed role of CaM as an integrator of different stress signaling pathways, which allows plants to maintain homeostasis between different cellular processes. In this review, we have attempted to present the current state of understanding of the role of CaM in modulating different stress-regulated proteins and its implications in augmenting abiotic stress tolerance in plants.

Expression of a cyclophilin OsCyp2-P isolated from a salt-tolerant landrace of rice in tobacco alleviates stress via ion homeostasis and limiting ROS accumulation

Cyclophilins are a set of ubiquitous proteins present in all subcellular compartments, involved in a wide variety of cellular processes. Comparative bioinformatics analysis of the rice and Arabidopsis genomes led us to identify novel putative cyclophilin gene family members in both the genomes not reported previously. We grouped cyclophilin members with similar molecular weight and subtypes together in the phylogenetic tree which indicated their co-evolution in rice and Arabidopsis. We also characterized a rice cyclophilin gene, OsCyp2-P (Os02g0121300), isolated from a salinity-tolerant landrace, Pokkali. Publicly available massively parallel signature sequencing (MPSS) and microarray data, besides our quantitative real time PCR (qRT-PCR) data suggest that transcript abundance of OsCyp2-P is regulated under different stress conditions in a developmental and organ specific manner. Ectopic expression of OsCyp2-P imparted multiple abiotic stress tolerance to transgenic tobacco plants as evidenced by higher root length, shoot length, chlorophyll content, and K+/Na+ ratio under stress conditions. Transgenic plants also showed reduced lipid peroxidase content, electrolyte leakage, and superoxide content under stress conditions suggesting better ion homeostasis than WT plants. Localization studies confirmed that OsCyp2-P is localized in both cytosol and nucleus, indicating its possible interaction with several other proteins. The overall results suggest the explicit role of OsCyp2-P in bestowing multiple abiotic stress tolerance at the whole plant level. OsCyp2-P operates via reactive oxygen species (ROS) scavenging and ion homeostasis and thus is a promising candidate gene for enhancing multiple abiotic stress tolerance in crop plants.