Flow cytometry as a method for assaying the biological activity of phytotoxins

A flow cytometric method has been developed for the qualitative and quantitative evaluation of the biological activities of phytotoxins from plant pathogenic fungi. The method utilized fresh wheat (Triticum aestivum L.) leaf protoplast preparations treated with purified phytotoxins, triticone A-B and triticone D. Subsequently, protoplasts were exposed to fluorescein diacetate, and analyzed by flow cytometry. Information acquired included fluorescence owing to esterase activity on fluorescein diacetate, and chlorophyll autofluorescence. Results indicate that triticone A-B has a rapid dose-dependent toxic effect on wheat protoplasts but triticone D has no toxic effect. This method can also yield information on the mechanism of action of phytotoxins that are relatively unstable or available only in small quantities.     

The identification and deletion of the polyketide synthase-nonribosomal peptide synthase gene responsible for the production of the phytotoxic triticone A/B in the wheat fungal pathogen Pyrenophora tritici-repentis

The economically important necrotrophic fungal pathogen, Pyrenophora tritici-repentis (Ptr), causes tan spot of wheat, a disease typified by foliar necrosis and chlorosis. The culture filtrate of an Australian Ptr isolate, M4, possesses phytotoxic activity and plant bioassay guided discovery led to the purification of necrosis inducing toxins called triticone A and B. High-resolution LC–MS/MS analysis of the culture filtrate identified an additional 37 triticone-like compounds. The biosynthetic gene cluster responsible for triticone production (the Ttc cluster) was identified and deletion of TtcA, a hybrid polyketide synthase (PKS)-nonribosomal peptide synthase (NRPS), abolished production of all triticones. The pathogenicity of mutant (ttcA) strains was not visibly affected in our assays. We hypothesize that triticones possess general antimicrobial activity important for competition in multi-microbial environments.     

Sustainable wheat (Triticum aestivum L.) production in saline fields: a review

Abstract

A large part of global agricultural fields, including the wheat (Triticum aestivum L.) ones, are subjected to various stresses including salinity. Given the increasing world population, finding methods and strategies that can alleviate salinity stress on crop yield production is of outmost importance. The presented review has consulted more than 400 articles related to the clean and sustainable production of wheat in saline fields affected by biological, environmental, economical, and social parameters including the important issue of climate change (global warming). The negative effects of salt stress on plant growth and the techniques, which have been so far detected to alleviate salinity stress on wheat growth have been analyzed and presented. The naturally tolerant species of wheat can use a range of mechanisms to alleviate salinity stress including sodium exclusion, potassium retention, and osmoregulation. However, the following can be considered as the most important techniques to enhance wheat tolerance under stress: (1) the biotechnological (crop breeding), biological (soil microbes), and biochemical (seed priming) methods, (2) the use of naturally tolerant genotypes, and (3) their combined use. The proper handling of irrigation water is also an important subject, which must be considered when planting wheat in saline fields. In conclusion, the sustainable and cleaner production of wheat under salt stress is determined by a combination of different parameters including the biotechnological techniques, which if handled properly, can enhance wheat production in saline fields.     

The PR-1a promoter contains a number of elements that bind GT-1-like nuclear factors with different affinity

A novel cDNA encoding for a peptidyl-prolyl-cis-trans-isomerase (PPIase) belonging to the FK506-binding protein (FKBP) family was isolated from wheat. It contains an open reading frame of 559 amino acids and it represents the first plant FKBP-PPIase to be cloned. It possesses a unique sequence which is composed of three FKPB-like domains, in addition to a putative tetratricopeptide repeat (TPR) motif and a calmodulin-binding site. The recombinant FKBP-PPIase expressed in and purified from Escherichia coli exhibits PPIase activity that is efficiently inhibited by the immunosuppressive drugs FK506 and rapamycin. Northern blot analysis showed that wheat FKBP was found mainly in young tissues. Polyclonal antibodies revealed the presence of cross-reacting proteins in embryos, roots and shoots. The unique structural features, the enzymatic activity and the presence of putative isoforms in wheat tissues indicate the possibility of the involvement of wheat PPIase in essential biological functions, similar to other members of the FKBP gene family.     

Static Magnetic Field Influence on the Activity of some Respiratory Enzymes in Wheat

Most of the published studies on magnetic field action on biological systems have examined reactions in animals, while a smaller number of studies have reported magnetic field effects in plants. The effects of static magnetic field on the activity of several key enzymes in plant metabolism, such as malate dehydrogenase, succinate oxidase, succinate dehydrogenase, and cytochrome oxidase in young wheat seedlings, have been investigated in this study. It appears that the observed changes in enzyme activity could be considered to be a result of the influence of the magnetic field on the reactivity of these enzymes, including effects on metal cations that regulate enzyme activity. The results support the idea of the existence of “biological windows,” particularly with respect to exposure time.     

Effects of plant growth regulators ambiol and phonk: biolphysical aspects

The antioxidative activity of two plant growth promoters, ambiol and phonk, in a model system of photo-induced glycyltriptophane oxidation was measured. It was shown that ambiol has a significant antioxidative activity, whereas phonk is a weak antioxidant. The effects of these compounds on DNA conformation were studied in vitro and in vivo (on wheat seed shoots). In vitro ambiol had a stronger effect as compared with phonk, whereas in vivo the latter produced a more essential effect than ambiol. The assumption was done that both compounds affect indirectly the genome expression activity. Possible mechanisms of biological activity of each compound are discussed.     

Biological activity of electrochemically activated solutions obtained in a diaphragm electrolyser

The biological activity of the catholyte and anolyte of bidistilled water in experiments with the germination of wheat grains in the period from March to May has been studied. The activity of solutions, which was characterized by the grain germination index, was high at the beginning of the period, then it gradually decreased and was equal to zero at the middle of the period; at the end of the period it gradually increased almost to initial values. It has been established that the effectiveness of bidistilled water anolyte was as a rule higher than that of catholyte throughout the observation period. At the beginning and end, the stimulating effect of anolyte was 5-5.5 times greater than that of catholyte. The seasonal changes in the biological activity of M 9 medium catholyte were compared with those of bidistilled water anolyte and catholyte. The stimulating effect of M 9 catholyte was estimated by changes in the growth of E. coli cells. The stimulating effect, which was estimated from an increase in the optical density of cell suspension in the initial period at a cultivation temperature of 20 degrees C was 55-60% relative to control (untreated medium). Then it decreased almost to zero in the middle of the period to increase again approximately to the initial values. The assumption has been made that the physicochemical causes of the influence of catholyte and anolyte of bidistilled water on wheat grains and of the culture medium catholyte on E. coli cells are of different nature.     

Investigation of the biological activity of solutions activated electrochemically in a membrane electrolyzer

The biological activity of the catholyte and anolyte of double distilled water was studied in experiments on the germination of wheat grains in the period from March to May. The activity of the solutions, which was characterized by a growth index, was high early in this period, then decreased almost to zero in the middle of the period, and then increased to about the initial value by the end of the period. Throughout, the efficiency of the anolyte of double distilled water generally exceeded the efficiency of the catholyte. Early and late in the period, the stimulatory effect of the anolyte exceeded that of the catholyte by a factor of 5–5.5. The changes in the biological activity of the catholyte and anolyte of double distilled water were also compared with the changes in the biological activity of the catholyte of nutrient medium M9. The stimulatory effect of the catholyte of the nutrient medium was evaluated from the change in the growth of E. coli cells. Early in the period at a cultivation temperature of 20°C, the stimulatory effect determined from the increase in the optical density of the cell suspension in the experiment with respect to a reference value was 55–60%. Next, the stimulatory effect decreased almost to zero in the middle of the period and increased to approximately initial value by the end of the period. It was assumed that the physicochemical mechanisms of action of the catholyte and anolyte of double distilled water on the wheat seed germination and of the catholyte of the nutrient medium on E. coli cell growth are of different nature.     

ABA metabolites induce group 3 LEA mRNA and inhibit germination in wheat

In plants, metabolism of the plant hormone (+)- S -abscisic acid (ABA) occurs by oxidation at the 8'-carbon on the ABA ring, producing (+)-8'-hydroxy-ABA (8'OH-ABA), which undergoes ring closure to phaseic acid (PA), or at the 7'-carbon producing 7'-hydroxy-ABA (7'OH-ABA). New methods for synthesixing and stabilizing 8'OH-ABA have enabled us for the first time to compare the biological activities of the ABA metabolites. 8'OH-ABA. PA and 7'OH-ABA. Activities were determined in wheat ( Triticum aestivum , cvs Brevor and Clarks Cream) using optically pure natural enantiomers of ABA. 8'OH-ABA, PA, and 7'OH-ABA. Comparison of wheat embryo germination inhibitory activity showed that only the metabolite 7'OH-ABA had significant activity (10 to 40% of effective ABA activity) as a germination inhibitor of wheat embryos. The metabolites had differential effects on ABA-responsive gene expression. Both 8'OH-ABA and PA were effective inducers of an ABA-responsive LEA (late embryogenesis abundant) gene, wheat group 3 LEA , in seedling roots, suggesting that ABA metabolites can maintain and prolong LEA gene expression. The metabolites had only a slight effect on accumulation of an ABA-responsive protein kinase ( PKABA1 ) mRNA or pMA1951 . These results are consistent with the idea that there are several control points in the ABA metabolic pathway and indicate that assessment of ABA signalling responses should include consideration of the biological activities of ABA metabolites.     

Purification of wheat germ RNA ligase. I. Characterization of a ligase-associated 5'-hydroxyl polynucleotide kinase activity

An RNA ligase that catalyzes the formation of a 2'-phosphomonoester-3',5'-phosphodiester bond in the presence of ATP and Mg2+ was purified approximately 6000-fold from raw wheat germ. A 5'-hydroxyl polynucleotide kinase activity copurified with RNA ligase through all chromatographic steps. Both activities cosedimented upon glycerol gradient centrifugation even in the presence of high salt and urea. RNA ligase and kinase activities sedimented as a single peak on glycerol gradients with a sedimentation coefficient of 6.2 S. The purified polynucleotide kinase activity required dithiothreitol and a divalent cation for activity and was inhibited by pyrophosphate and by ADP. The kinase phosphorylated a variety of 5'-hydroxyl-terminated polynucleotide chains including some that were substrates for the RNA ligase (e.g. 2',3'-cyclic phosphate-terminated poly(A)) and others that were not ligase substrates (e.g. DNA or RNA containing 3'-hydroxyl termini). RNA molecules containing either 5'-hydroxyl or 5'-phosphate and 2',3'-cyclic or 2'-phosphate termini were substrates for the purified RNA ligase activity. The rate of ligation of 5'-hydroxyl-terminated RNA chains was greater than that of 5'-phosphate-terminated molecules, suggesting that an interaction between the wheat germ kinase and ligase activities occurs during the course of ligation.     

干旱条件下小麦冠层温度及其性状的关联研究

通过对小麦冠层温度和有关性状的长期观测,发现自然界存在冠层温度持续偏低的小麦,在干旱条件下,它的叶片功能期、叶绿素含量、蒸腾速率、净光合速率、可溶性蛋白含量和超氧化物歧化酶活性等重要性状明显优于冠层温度持续偏高的小麦品种,且丙二醛(MDA)积累速度慢,在籽粒灌浆期表现尤为明显。冷型小麦表现出干旱胁迫下仍然具有代谢功能较好、活力旺盛和抗早衰能力较强的特征,这进一步拓展了冷型小麦的应用范围,对于加速适应于干旱条件的优良品种选育并将其推向生产具有十分重要的意义。     

植物光效生态学研究 Ⅰ.小麦光合作用午休的原因

本文报道了用小麦作材料,从作物生理生态特性方面研究了小麦光合作用午休的原因。试验结果证明:在中午前后,小麦叶片气孔开关数目与光合作用午休密切相关;大部分小麦光合作用午休与生物节律无关;中午遮光后,光合作用加强和生物学产量提高,说明光合产物的累积是形成光合作用午休的重要因素之一。     

Beta-adenosine, a bioactive compound in grass chaff stimulating mushroom production

Fructification and yield of the edible mushrooms Pleurotus pulmonarius and Stropharia rugosoannulata are clearly enhanced when wheat straw is supplemented with 30% Lolium perenne grass chaff. The bioactive compound in the methanol extract of grass chaff was identified as beta-adenosine. In vitro biological activity tests showed that 0.012 mg of beta-adenosine per ml of medium stimulated earlier fructification of Pleurotus pulmonarius. Mushroom fruiting trials showed that when 12 mg beta-adenosine was added to 1 kg wet wheat straw, primordia of Pleurotus pulmonarius appeared two days earlier and primordia of Stropharia rugosoannulata appeared 18 days earlier when compared to pure wheat straw substrate. This concentration of beta-adenosine had no impact on the mushroom yield of Pleurotus, but resulted in a 2.2 fold increase in yield for Stropharia. beta-Adenosine at 25 mg per kg wet wheat straw increased the yield of Pleurotus with 52% and the yield of Stropharia with 258%, but this concentration delayed primordial formation in Pleurotus.     

小麦原生质体高效转化体系的建立

植物原生质体广泛应用于植物基因功能研究中,包括瞬时基因表达、亚细胞定位、蛋白互作和蛋白活性分析等。当前,小麦基因的亚细胞定位和功能分析,大多利用模式植物拟南芥等异源的原生质体,易于造成研究结果的不准确。为避免这种情况,小麦原生质体制备及高效转化体系的建立与应用是必需的。在PEG介导的小麦原生质体转化过程中,原生质体分泌的核酸酶大量降解质粒DNA,转化效率的提高因此受到阻碍。为了建立小麦原生质体的高效转化体系,本文测试了抑制胞外核酸酶活性的因素和提高质粒DNA浓度等多个条件对转化效率的影响。结果表明,转化过程中加入双倍用量的质粒DNA进行转化,且始终保持低温环境(1℃)用以抑制核酸酶酶活性,可以使小麦原生质体的转化效率提高至85%。本文还将该系统成功地应用于2个小麦抗病相关蛋白的亚细胞定位研究,证明了该系统的高效性和实用性。该研究对未来相关研究有一定参考价值。     

冷型小麦对干旱和阴雨的双重适应性

通过 10余年的研究 ,发现冠层温度持续偏低的冷型小麦具有广幅生态适应性 ,即在正常天气条件下 ,它的活力较好、代谢功能较强、具有较好的抗早衰能力 ,优于其它类型小麦 ;而在气象要素反差很大的干旱和连阴雨天气下 ,冷型小麦和暖型小麦、中间型小麦相比 ,不但冠层温度依然持续偏低 ,且在叶片功能期、叶绿素含量、可溶性蛋白质含量、过氧化氢酶活性、净光合速率和籽粒饱满指数等一系列重要内、外性状上仍能继续保持优势。这种对干旱和阴雨条件的双重适应性是冷型小麦一种宝贵的生物学品质 ,它有可能为小麦在多种气象生态条件下的高产、稳产打下较为坚实的生态生理基础 ,并能为优良品种的选育提出一个把冷温特征作为重要内容的较为新鲜的育种目标。     

Correlation between Plant Growth Regulator Release Rate and Bioactivity for the Series of Newly Synthesized Phytoactive Polymers

Phytoactive polymers are high molecular weight systems in which a plant growth regulator (PGR) unit is attached to the polymeric chain by a hydrolyzable chemical bond. The release rate of the PGR is linked to the biological activity of the phytoactive polymer and can be controlled by properties inherent in the whole macromolecular system. In this study the correlation of biological activity and plant growth regulator hydrolytic release rate was investigated for the series of newly synthesized 2,4-dichlorophenoxyacetic acid (2,4-D) polymeric esters. The polymers synthesized differ in their molecular weight, side group structure, and 2,4-D residue content. The influence of these polymer characteristics on the 2,4-D hydrolytic release was investigated, and it was demonstrated that hydrolysis rate substantially depends on the polymer molecular weight, side group structure, and 2,4-D residue content. It was also demonstrated that phytoactive polymer bioactivity depends on the hydrolysis rate of the polymers, and in dependence of this parameter can provide stimulating or inhibiting activity. Biological activity was illustrated by the elongation of wheat and barley coleoptiles.     

A wheat germ cell-free system is a novel way to screen protein folding and function

For high-throughput protein structural analysis, it is indispensable to develop a reliable protein overexpression system. Although many protein overexpression systems, such as that involving Escherichia coli cells, have been developed, the number of overexpressed proteins showing the same biological activities as those of the native proteins is limited. A novel wheat germ cell-free protein synthesis system was developed recently, and most of the proteins functioning in solution were synthesized as soluble forms. This suggests the applicability of this protein synthesis method to determination of the solution structures of functional proteins. To examine this possibility, we have synthesized two (15)N-labeled proteins and obtained (1)H-(15)N HSQC spectra for them. The structural analysis of these proteins has already progressed with an E. coli overexpression system, and (1)H-(15)N HSQC spectra for biologically active proteins have already been obtained. Comparing the spectra, we have shown that proteins synthesized with a wheat germ cell-free system have the proper protein folding and enough biological activity. This is the first experimental evidence of the applicability of the wheat germ cell-free protein synthesis system to high-throughput protein structural analysis.     

Glycoproteins from Russian wheat aphid infested wheat induce defence responses

Elicitors are molecules which can induce the activation of plant defence responses. Elicitor activity of intercellular wash fluid from Russian wheat aphid, Diuraphis noxia (Mordvilko) infested resistant (cv Tugela DN), and susceptible (cv Tugela), wheat (Triticum aestivum L.), was investigated. Known Russian wheat aphid resistance related responses such as peroxidase and beta-1,3-glucanase activities were used as parameters of elicitor activity. The intercellular wash fluid from infested resistant plants contains high elicitor activity while that from infested susceptible plants contains no or very little elicitor activity. After applying C-18 reverse phase and concanavalin A Sepharose chromatography, elicitor active glycoproteins were isolated from the intercellular wash fluid of Russian wheat aphid infested resistant wheat. The elicitor-active glycoproteins separated into three polypeptides during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isolated glycoproteins elicited peroxidase activity to higher levels in resistant than in susceptible cultivars. It was evident that the glycoproteins were probably a general elicitor of plant origin. Information gained from these studies is valuable for the development of plant activators to enhance the defence responses of plants.     

Purification and characterization of a phosphotyrosyl-protein phosphatase from wheat seedlings

A neutral phosphatase which catalyzes the hydrolysis of p-nitrophenylphosphate has been purified to homogeneity from wheat seedlings. The enzyme is a monomeric glycoprotein exhibiting a molecular weight of 35,000, frictional ratio of 1.22, Stokes' radius of 260 nm, and sedimentation coefficient of 3.2 S. That the enzyme is a glycoprotein is surmised from its chromatographic property on Concanavalin A-Sepharose column. An examination of the substrate specificity indicates that the enzyme exhibits a preference for phosphotyrosine over a number of phosphocompounds, including p-nitrophenylphosphate and several glycolytic intermediates. Both phosphoserine and phosphothreonine are not hydrolyzed by the enzyme. The phosphatase activity is not affected by high concentrations of chelating agents and does not require metal ions. Molybdate, orthovanadate, Zn2+, and Hg2+ are all potent inhibitors of the phosphatase activity. The ability of the phosphatase to dephosphorylate protein phosphotyrosine has been investigated. [32P-Tyr]poly(Glu,Tyr)n, [32P-Tyr]alkylated bovine serum albumin, [32P-Tyr]angiotensin-I, and [32P-Tyr]band 3 (from human erythrocyte) are all substrates of the phosphatase. On the other hand, the enzyme has no activity toward protein phosphoserine and phosphothreonine. Our result further indicates that the neutral phosphatase is distinct from the wheat germ acid phosphatase. The latter enzyme is found to dephosphorylate phosphotyrosyl as well as phosphoseryl and phosphothreonyl groups in proteins. In light of the many similarities in properties to phosphotyrosyl protein phosphatases isolated from several sources, it is suggested that the wheat seedling phosphatase may participate in cellular regulation involving protein tyrosine phosphorylation.     

Molecular mapping of a non-host resistance gene YrpstY1 in barley (Hordeum vulgare L.) for resistance to wheat stripe rust

Cultivated barley (Hordeum vulgare L.) is considered as a non-host or inappropriate host species for wheat stripe rust caused by Puccinia striiformis f. sp. tritici. Most barley cultivars show a broad-spectrum resistance to wheat stripe rust. To determine the genes for resistance to wheat stripe rust in barley, a cross was made between a resistant barley line Y12 and a susceptible line Y16. The two parents, F(1) and 147 BC(1) plants were tested at seedling stage with Chinese prevalent race CYR32 of Puccinia striiformis f. sp. tritici by artificial inoculation in greenhouse. The results indicated that Y12 possessed one dominant resistance gene to wheat stripe rust, designated YrpstY1 provisionally. A total of 388 simple sequence repeat (SSR) markers were used to map the resistance gene in Y12 using bulked segregant analysis. A linkage map, including nine SSR loci on chromosome 7H and YrpstY1, was constructed using the BC(1) population, indicating that the resistance gene YrpstY1 is located on chromosome 7H. It is potential to transfer the resistance gene into common wheat for stripe rust resistance.