The distribution of serine proteinase inhibitors in seeds of the Asteridae.
Alexander V. Konarev
All-Russian Institute of Plant Protection (VIZR), Podbelsky 3, St. Petersburg 196608, Russia
Jonathan Griffin
Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ , UK
Galina Yu. Konechnaya
V.L. Komarov Botanical Institute, Prof. Popova 2, St. Petersburg 197376, Russia
Peter R. Shewry
Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ , UK
The Asteridae is one of the most successful clades of flowering plants comprising some 80,000 species. Despite this diversity, analysis of seeds from 398 species (representing 8 orders, 32 families and 181 genera) showed just two major types of serine proteinase inhibitors (PI). PIs of the potato inhibitor I family were widely distributed. These had M(r) of 7000-7500 and were inhibitory to subtilisin and one or more other proteinases (but only rarely elastase). The second major group was TI related to the well-characterised Bowman-Birk inhibitors of legume seeds but these varied widely in their sequences and structure. In addition to these two groups of inhibitors, seeds of the Solanaceae also often contained PI of the potato inhibitor II family while some other asterids contained inhibitors whose relationships were not established.
I.N. Anisimova
Institute of Plant Industry (VIR), Bolshaya Morskaya 44, St. Petersburg , 190000, Russia
Al.V. Konarev
All-Russian Institute of Plant Protection (VIZR), Podbelsky 3, St. Petersburg , 189620, Russia
V.A. Gavrilova
Institute of Plant Industry (VIR), Bolshaya Morskaya 44, St. Petersburg , 190000, Russia
V.T. Rozhkova
Institute of Plant Industry (VIR), Bolshaya Morskaya 44, St. Petersburg , 190000, Russia
R.F. Fido
Long Ashton Research Station, University of Bristol , Long Ashton, Bristol BS41 9AF , U.K.
A.S. Tatham
Long Ashton Research Station, University of Bristol , Long Ashton, Bristol BS41 9AF , U.K.
P.R. Shewry
Long Ashton Research Station, University of Bristol , Long Ashton, Bristol BS41 9AF , U.K.
Abstract
2S albumin fractions were isolated by a modified acetone precipitation method (Kortt and Caldwell 1990) from seeds of 103 sunflower ( Helianthus annuus L.) accessions and analysed by SDS-PAGE, IEF and RP-HPLC. Two methionine-rich albumins SFA7 and SFA8 showed no differences in mobility on SDS-PAGE gels but were readily separated by RP-HPLC. Their levels also varied widely between different genotypes, in relation to each other and as proportions of the total albumin fraction. A variant form of SFA8 was identified which differed from the normal SFA8 in its pI (6.5 compared to 6.0) and mobility on SDS-PAGE. N -terminal sequences of both the variant form of SFA8 and the major form of SFA7 were identical to that reported previously for the normal form of SFA8 from the cultivar Hysun (Kortt et al., 1991) indicating their structural relatedness. Analysis of segregation in the F 2 of the cross between lines VIR130 (variant SFA8) and VIR104 (normal SFA8) showed that the normal and variant forms of SFA8 are encoded by alleles at a single Mendelian locus. The levels of SFA7 and SFA8 in the seeds of parental lines, F 1 hybrids and individual F 2 seeds classified from SDS-PAGE and IEF as homozygous for normal SFA8 (VIR104 type), homozygous for variant SFA8 (VIR130 type) and heterozygous (F 1 type) were determined by RP-HPLC. Seeds of the parental line VIR130 contained 3.7% SFA7 and 19.0% SFA8 whereas seeds of VIR104 contained 9.9% SFA7 and 12.8% SFA8. The F 1 hybrid seeds contained a higher total amount of SFA7+8 proteins (32% comparing to 22% in each parent) which was largely accounted for by a high proportion of SFA7. The mean combined proportions of SFA7+8 in each of the three phenotypic classes of F 2 seeds were about 1819% of the total. However, the combined proportions of SFA7+8 varied in the range 1020% among the individual seeds. The ratio of SFA7 to SFA8 was highest in the VIR104-type and heterozygous seeds, with the amount of SFA7 exceeding that of SFA8 in six heterozygous seeds. The proportions of SFA7 and SFA8 were inversely correlated among individual F 2 seeds. The results suggest that the amounts and proportions of SFA7 and SFA8 are determined by genetic factors in addition to availability of sulphur.
Keywords
genetic control, nutritional quality, seed proteins, sunflower
XI-th Int.Congress on Plant-Microbe Interactions, July 18-26, 2003, St.Petersburg, Russia
Konarev Al.V 1 ., Kochetkov V.V. 2 , Shewry P.R. 3
1 All-Russia Research Institute for Plant Protection. Podbelsky sh., 3, Pushkin-8, 196608, St.-Petersburg , Russia . e-mail: al_konarev@hotmail.com. 2 All-Russia Research Institute for Agricultural Microbiology (address as for 1 ). 3 Rothamsted Research, Harpenden, Herts AL5 2JQ , UK . e-mail: peter.shewry@bbsrc.ac.uk.
Proteinase inhibitors present in seeds and vegetative parts of plants are of interest as antifungal, antiviral and insecticidal agents. Extracellular serine proteinases are characteristic of many pathogenic fungi including subtilisin-like enzymes in Sclerotinia slerotiorum and Colletotricum lindemuthianum and trypsin-like enzymes in other species. Seeds of about 460 species representing 235 genera of 32 families from 8 orders of Asteridae (Asterales, Apiales, Dipsacales, Lamiales, Solanales, Gentianales, Ericales and Cornales) were initially screened for inhibitors (I) of four serine proteinases: subtilisin (S), trypsin (T), chymotrypsin (C) and elastase (E). Subtilisin inhibitors with M r about 7000-7500, which were also active against one or more other serine proteinases, appeared to be the most widely distributed inhibitor type and, according to protein sequencing data, were related to the potato I inhibitor family. T/SIs and C/SIs were found in many Compositae (sunflower, safflower, Cosmos ), Apiaceae (carrot, dill), Pedaliaceae (sesame), and Ericaceae ( Vaccinum species) while E/SIs were more rare (Apiaceae, Anthriscus silvestris ). TIs showed the greatest variation in M r and properties. Several unique low-molecular TIs were found in Asteridae, including a cyclic SFTI-1 with M r 1514 in sunflower (Konarev et al., 1999; Luckett et al., 1999) and a TI with M r about 3900 in Veronica species. The inhibitors identified may have applications in developing plant varieties which are resistant to fungi and other pests and pathogens.
Cross analysis of the interaction of alpha-amylase and proteinase components of insects with protein[aceous] inhibitors from wheat endosperm
Al. V. Konarev & Yu.V.Fomicheva
All-Russian Institute of Plant Protection (VIZR), Podbelsky 3, St. Petersburg 196608, Russia
The components of the digestive proteinases that hydrolyze gelatin, N[alpha]-benzoyl-DL-arginine-p-nitroanilide (BAPNA), acetyl-DL-phenylalanine-2-naphthyl ester (APNE), as well as alpha-amylases, were studied on eight species of insects that damage cereal grains or grain products, by isofocusing (IEF) in PAAG. Novel sensitive modification of method was developed for detection of insect trypsin-like proteinases after IEF including transfer proteinases from separating gel to nitrocellulose membrane and using of BAPNA followed by strengthening and stabilization of colored zones of proteinase activity by the reaction of diazotization of released nitroaniline. New “cross” methods were used to analyse the interaction of the IEF bands (components) of alpha-amylases and proteinases with IEF bands of proteinaceous inhibitors from wheat endosperm. It was shown that the components of insect proteinase complexes are characterized by pronounced species specificity. The components of the spectra differ in their ability to hydrolyze proteinaceous and synthetic substrates and with respect to trypsin, chymotrypsin, and cysteine (thiol) proteinase inhibitors. The information obtained by the methods proposed can be used for a preliminary “classification” of the individual enzyme fractions before their detailed investigations, as well as in an assessment of the possible protective role of inhibitors in connection with the peculiarities of complexes of digestive hydrolases in various species of insects.
Insect species: Tenebrio molitor L., Rhysoperta dominica F. (Rhizopertha dominica), Oryzaephilus surinamensis L., Tribolium confusum Duv., Tribolium castaneum Herbst., Sitophilus granarius L., Sitophilus oryzae L., Eurygaster integriceps Put.
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