Bioevaluation of Subabul (Leucaena leucocephala) proteinase inhibitors on Helicoverpa armigera

Helicoverpa armigera, a highly polyphagous pest, has a broad host spectrum, causes significant levels of yield loss in many agriculturally important crops. Serine primarily responsible for most of the proteolytic activity in the larval gut of lepidopteron insects. Neonate larvae were reared on artificial diet and chickpea seeds smeared with Subabul Trypsin Inhibitor. Larvae fed with artificial diet showed reduction in larval weight up to 21% (HSTI) and 43% (LSTI). However, larvae fed on seeds showed significant reduction in weight, 52.4% (HSTI) and 60.3% (LSTI), suggesting that the diet also plays a vital role on the effectiveness of the inhibitors on larval growth and development. HSTI and LSTI inhibited the gut proteinases from larvae fed on artificial diet significantly (41.40% and 64.36%) compared to the gut proteinases (27.80% and 38.90%) from larvae fed on chickpea seeds. Seeds smeared with 10,000 TIU resulted in complete mortality of larvae while there was no mortality observed in artificial diet. The results reveal that LSTI is a stronger inhibitor of insect gut proteinases and for larvae fed with poor nutrition in the natural ecosystems, low level expression of inhibitor would be enough to affect the growth and development. Handling editor: Chen-Zhu Wang     

Characterization of two midgut proteinases of Helicoverpa armigera and their interaction with proteinase inhibitors

Two serine proteinases from the midgut of Helicoverpa armigera have been partially purified and characterized. One proteinase, HGP-1, was capable of hydrolyzing a synthetic substrate of elastase and was inhibited by elastatinal. The second proteinase, HGP-2, was inhibited by a trypsin inhibitor. Molecular weights of HGP-1 and HGP-2 were approximately 26.0 and 29.0kDa, respectively. Both the proteinases exhibited alkaline pH optima in the range of 10-11. Furthermore, interaction of HGP-1 and HGP-2 with proteinase inhibitors (PIs) from host and non-host plants was studied. HGP-1 was not only insensitive to a PI from chickpea (host) but was also able to degrade it. The same PI from chickpea was able to inhibit over 50% activity of HGP-2. On the contrary, PIs from potato (non-host) showed strong inhibition of both, HGP-1 and HGP-2 and also demonstrated protection of chickpea seed proteins from digestion by both the HGPs. These results could provide important clues in designing strategies for sustainable use of plant PIs in developing insect-tolerant transgenic plants.     

Bitter gourd proteinase inhibitors: potential growth inhibitors of Helicoverpa armigera and Spodoptera litura

Proteinase inhibitors (PIs) from the seeds of bitter gourd (Momordica charantia L.) were identified as strong inhibitors of Helicoverpa armigera gut proteinases (HGP). Biochemical investigations showed that bitter gourd PIs (BGPIs) inhibited more than 80% HGP activity. Electrophoretic analysis revealed the presence of two major proteins (BGPI-1 and-2) and two minor proteins (BGPI-3 and-4) having inhibitory activity against both trypsin and HGP. The major isoforms BGPI-1 and BGPI-2 have molecular mass of 3.5 and 3.0 kDa, respectively. BGPIs inhibited HGP activity of larvae fed on different host plants, on artificial diet with or without added PIs and proteinases excreted in fecal matter. Degradation of BGPI-1 by HGP showed direct correlation with accumulation of BGPI-2-like peptide, which remained stable and active against high concentrations of HGP up to 3 h. Chemical inhibitors of serine proteinases offered partial protection to BGPI-1 from degradation by HGP, suggesting that trypsin and chymotrypsin like proteinases are involved in degradation of BGPI-1. In larval feeding studies, BGPIs were found to retard growth and development of two lepidopteran pests namely Helicoverpa armigera and Spodoptera litura. This is the first report showing that BGPIs mediated inhibition of insect gut proteinases directly affects fertility and fecundity of both H. armigera and S. litura. The results advocate use of BGPIs to introduce insect resistance in otherwise susceptible plants.     

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.     

Identification of potent inhibitors of Helicoverpa armigera gut proteinases from winged bean seeds

Dry mature seeds of winged bean (Psophocarpus tetragonolobus L., DC.) (WB) contain several proteinase inhibitors. Two-dimensional gel analysis of WB seed protein followed by activity visualization using a gel-X-ray film contact print technique revealed at least 14 trypsin inhibitors (TIs) in the range of 28-6 kD. A total of seven inhibitors (WBTI-1 to 7) were purified by heat treatment and gel filtration followed by elution from preparative native gels. Based on their biochemical characterization such as molecular mass, pI, heat stability, and susceptibility to inactivation by reducing agents, WBTI-1 to 4 are Kunitz type inhibitors while WBTI-5 to 7 are classified as Bowman-Birk type serine proteinase inhibitors. Although Kunitz type TIs (20-24 kD) of WB have been reported, the smaller TIs that belong to the Bowman-Birk type have not been previously characterized. Seven major TIs isolated from WB seed were individually assessed for their potential to inhibit the gut proteinases (HGP) of Helicoverpa armigera, a pest of several economically important crops, which produces at least six major and several minor trypsin/chymotrypsin/elastase-like serine proteinases in the gut. WBTI-1 (28 kD) was identified as a potent inhibitor of HGP relative to trypsin and among the other WBTIs; it inhibited 94% of HGP activity while at the same concentration it inhibited only 22% of trypsin activity. WBTI-2 (24 kD) and WBTI-4 (20 kD) inhibited HGP activity greater than 85%. WBTI-3,-5,-6 and-7 showed limited inhibition of HGP as compared with trypsin. These results indicate that WBTIs have different binding potentials towards HGP although most of the HGP activity is trypsin-like. We also developed a simple and versatile method for identifying and purifying proteinase inhibitors after two-dimensional separation using the gel-X-ray film contact print technique.     

Effect of salinity on tomato (Lycopersicon esculentum Mill.) during seed germination stage

A study was conducted using ten genetically diverse genotypes along with their 45F₁ (generated by diallel mating) under normal and salt stress conditions. Although, tomato (Lycopersicon esculentum Mill.) is moderately sensitive to salinity but more attention to salinity is yet to be required in the production of tomato. In present study, germination rate, speed of germination, dry weight ratio and Na⁺/K⁺ ratio in root and shoot, were the parameters assayed on three salinity levels; control, 1.0 % NaCl and 3.0 % NaCl with Hoagland’s solution. Increasing salt stress negatively affected growth and development of tomato. When salt concentration increased, germination of tomato seed was reduced and the time needed to complete germination lengthened, root/shoot dry weight ratio was higher and Na⁺ content increased but K⁺ content decreased. Among the varieties, Sel-7 followed by Arka Vikas and crosses involving them as a parent were found to be the more tolerant genotypes in the present study on the basis of studied parameters.     

NMR characterization of endogenously O-acetylated oligosaccharides isolated from tomato (Lycopersicon esculentum) xyloglucan

Eight oligosaccharide subunits, generated by endoglucanase treatment of the plant polysaccharide xyloglucan isolated from the culture filtrate of suspension-cultured tomato (Lycopersicon esculentum) cells, were structurally characterized by NMR spectroscopy. These oligosaccharides, which contain up to three endogenous O-acetyl substituents, consist of a cellotetraose core with alpha-D-Xylp residues at O-6 of the two beta-D-Glcp residues at the non-reducing end of the core. Some of the alpha-D-Xylp residues themselves bear either an alpha-L-Arap or a beta-D-Galp residue at O-2. O-Acetyl substituents are located at O-6 of the unbranched (internal) beta-D-Glcp residue, O-6 of the terminal beta-D-Galp residue, and/or at O-5 of the terminal alpha-L-Arap residue. Structural assignments were facilitated by long-range scalar coupling interactions observed in the high-resolution gCOSY spectra of the oligosaccharides. The presence of five-bond scalar coupling constants in the gCOSY spectra provides a direct method of assigning O-acetylation sites, which may prove generally useful in the analysis of O-acylated glycans. Spectral assignment of these endogenously O-acetylated oligosaccharides makes it possible to deduce correlations between their structural features and the chemical shifts of diagnostic resonances in their NMR spectra.     

Oviposition deterrents in larval frass of the cotton boll worm, Helicoverpa armigera (Lepidoptera: Noctuidae): chemical identification and electroantennography analysis

Oviposition deterrents in the frass of cotton bollworm (CBW), Helicoverpa armigera larvae fed on an artificial diet (FA) and on cotton Gossypium hirsutum leaves (FC) were investigated by behavioral bioassays and electroantennography analyses in the laboratory. It was found that a water suspension or a hexane extract of the frass FA or FC, in contrast to the corresponding foods, significantly deterred oviposition of conspecifics. When hexane extracts of the frass FA and FC were further partitioned into polar and neutral lipid fractions, two polar fractions significantly reduced oviposition. The neutral fraction from frass FC also exhibited significant deterrence, although the activity was much lower than that of the corresponding polar fraction. The polar lipid fractions contained several fatty acids, mainly palmitic and oleic acid at the ratio nearly 1:1. A blend of authentic fatty acids of the same composition found in frass FA or FC mimicked the deterring effect. Moreover, these fatty acids and their blend at the ratio found in frass FA or FC elicited significant electroantennogram responses and typical dose-response curves. Thus, it is suggested that CBW larvae may deploy two types of oviposition deterrents: a non-specific and a specific one. The former is a blend of fatty acids, independent of food and plays an important role in oviposition deterrence, whereas the latter may be produced only when the larvae feed on cotton leaves. The possible explanations of this deployment have also been discussed.     

外源NO与蔗糖对盐胁迫下番茄(Lycopersicon esculentum Mill)幼苗氧化损伤的保护效应

选取长至6～8片真叶的健康番茄(Lycopersicon esculentum Mill)幼苗,分别进行蔗糖、硝普钠(sodium nitropresside, SNP, 作为外源NO供体)及其体积比例组合(1:1)处理;36h后施以NaCl胁迫,并分别于0h(胁迫前)、24h、48h和72h 取样,进行相关生理生化指标测定.具体5个实验处理如下:A. 蒸馏水(CK); B. 100 mmol/L NaCl;C. 0.1 mmol/L SNP 100 mmol/L NaCl;D. 0.1 mmol/L SNP 1.0mmol/L蔗糖 100 mmol/L NaCl; E. 1.0 mmol/L蔗糖 100 mmol/L NaCl.结果表明:与SNP和蔗糖单独处理相比,二者组合处理对缓解盐胁迫下番茄幼苗的氧化损伤存在正协同效应,主要表现在进一步增强了番茄幼苗超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)、抗坏血酸过氧化物酶(APX)和谷胱甘肽还原酶(GR)的活性;提高了脯氨酸(Pro)的含量,同时膜脂过氧化产物丙二醛(MDA)含量显著降低(P＜0.05).采用聚丙烯酰胺浓度梯度凝胶电泳对盐胁迫24 h和48 h材料的POD同功酶检测表明,当NaCl单独处理时,番茄幼苗叶片POD同功酶第V条带缺失,其它谱带酶量减少,抑制了POD同功酶的表达;SNP和蔗糖单独处理能够保护盐胁迫(24、48h)所导致的POD同功酶条带的完整;而组合处理既保证了POD同功酶条带的完整,又加强了酶量的表达.随着盐胁迫时间的延长,其氧化损伤程度愈烈,SNP和蔗糖组合处理能够更有效地缓解盐胁迫对番茄幼苗植株造成的氧化损伤.