Generation of transgenic wheat (Triticum aestivum L.) for constitutive accumulation of an Aspergillus phytase

The Aspergillus niger phytase-encoding gene (phyA) has been constitutively expressed in wheat. Transgenic wheat lines were generated by microprojectile bombardment of immature embryos, using the bar-Bialaphos selection system. The bar and the phyA gene expression were controlled by the maize ubiquitin-1 promoter. To ensure secretion and glycosylation of the microbial phytase, an expression cassette was designed (Ubi-SP-Phy) where an -amylase signal peptide sequence was inserted between the promoter and the phytase coding region. A similar cassette was constructed without the signal peptide sequence (Ubi-Phy). Five lines of fertile wheat transformed with the Ubi-SP-Phy were generated and two lines with the Ubi-Phy construct. The inheritance of the phyA gene was monitored through three generations. Western blotting of leaf and seed derived protein revealed the presence of an immunoreacting polypeptide of the size expected for the Aspergillus phytase. Up to 25 days after pollination, the heterologous phytase was exclusively present in the pericarp-seed coat-aleurone fraction. Thereafter, it accumulated in the endosperm in amounts exceeding that found in the seed coat and aleurone. The phyA mRNA and derived protein could at no stage be detected in the embryo. The Ubi-SP-Phy transgenic seeds exhibited up to 4-fold increase of phytase activity while up to 56% increase was found in Ubi-Phy plants. It is concluded that a functional Aspergillus phytase can be produced in significant amounts in wheat grains. This may be of relevance for improving the phytate-phosphorus digestibility when wheat grains are used for non-ruminant animal feed.     

Concerted Action of Endogenous and Heterologous Phytase on Phytic Acid Degradation in Seed of Transgenic Wheat (Triticum aestivum L.)

Expression of heterologous phytases in crops offers a great potential for improving phosphate and mineral bioavailability in food and feed. In this context it is of relevance to describe the concerted action of endogenous and hetrologous phytases on the transgenic seed inositol phosphate profile. Here we report metal-dye detection HPLC analysis of inositol phosphate degradation in flour from transgenic wheat materials possessing wheat endogenous 6-phytase [EC 3.1.3.26] and Aspergillus 3-phytase [EC 3.1.3.8] activities under the control of the maize ubiquitin-1 promoter and the wheat high molecular weight glutenin subunit 1DX5 promoter respectively. During 50 min incubation there is an accumulation of InsP5 to InsP2 breakdown products in non-transgenic material. Aspergillus niger phytase specific breakdown products are transiently detected in transgenic material but after 50 min incubation virtually all InsP5, InsP4 and InsP3 isomers are hydrolysed.     

Gibberellic acid (GA) increases fibre cell differentiation and secondary cell-wall deposition in spring wheat (Triticum aestivum L.) culms

The role of gibberellic acid (GA) in differentiation and secondary cell-wall deposition of fibre cells of spring wheat (Triticum aestivum) culms was studied using applications of GA and chlormequat (a GA biosynthesis inhibitor). In certain genotypes, higher GA levels may increase the number of cortical fibre cell files by changing cell fate from parenchyma to fibre, and induce thicker secondary cell-walls.     

外源水杨酸对光抑制条件下小麦叶片光合作用的影响

以浓度为50、100、200 mg·kg-1的水杨酸(SA)预先处理灌浆期的小麦叶片,可有效防护强光所致的氧化损伤,维持较高的超氧化物歧化酶(SOD)和抗坏血酸过氧化物酶(APX)活性,减轻丙二醛(MDA)积累.叶片在光抑制条件下,可维持较高的通过PSⅡ电子传递速率(Fm/Fo)、PSⅡ原初光化学效率(Fv/Fm)、PSⅡ量子效率(ΦPSⅡ)、光化学猝灭系数(qP)、净光合速率(Pn)和较低的非光化学猝灭系数(qNP).其中,以较低浓度SA(50 mg·kg-1)的效果较好.     

Endogenous gibberellins in young seedlings of wheat (Triticum aestivum) cultivars

Gibberellins A₁, A₃, A₄ and A₇ were identified by combined gas chromatography mass spectrometry (GC-MS) in leaf and stem tissues of 17-day-old seedlings of wheat ( Triticum aestivum L. ), cvs Siete Cerros (semi-dwarf, Rht1) and Møystad (tall), of F₁, hybrids from the cross Møystad × Siete Cerros and of 2 selected lines from the cross Møystad x Sonora 64 (Rht1 and Rht2). GA, and GA, were identified by full scan mass spectra separately in all 5 extracts, GA₄ and GA₇, were identified by selected ion monitoring in a bulked fraction. About 90% of the biological activity (Tan-ginbozu dwarf rice bioassay) in all 5 extracts was due to the GA₁/GA₃-fraction.     

Preferential utilization of organic and inorganic sources of phosphorus by wheat plant

On soils of low P supply organic P (Po) makes up a similar or even larger part in soil solution than inorganic P (Pi). The ability of wheat (Triticum aestivum L., cv. Star) plants to hydrolyze and absorb this Po in comparison to similar concentrations of Pi was studied. Four concentration levels of Pi and Po were obtained by extracting two soils with deionized water in a ratio of 1:1 and concentrating the resulting filtrate by freeze drying to different degrees. The concentration of Pi varied between 5 and 36 μM and Po between 3 and 22 μM. Wheat seedlings were grown in these solutions for 12 and 24 h and acid and alkaline phosphatase activity determined. The reduction of Po concentration in solution expressed on a root length basis gave the rate of Po hydrolysis and the reduction in concentration of Pi and Po gave the P inflow into the roots. No alkaline phosphatase activity was detected. The activity of wheat root acid phosphatase increased with Po concentration in solution. Phosphorus uptake was 2 to 6 fold higher from Pi than from Po at similar concentrations of both. The rate of uptake from Pi, the inflow, as well as the rate of hydrolysis of Po increased linearly with concentration but at similar concentration the inflow was 2 to 4 times higher than the rate of Po hydrolysis. Results suggest that plants can utilize Po after hydrolysis by phosphatase, but Pi is more important and preferentially used by plants; Po may be essential for plant nutrition especially in high P-fixing soils.     

Salicylic acid changes the properties of extracellular peroxidase activity secreted from wounded wheat (Triticum aestivum L.) roots

Summary.  Wheat (Triticum aestivum L.) roots released proteins showing peroxidase activity in the apoplastic solution in response to wound stress. Preincubation of excised roots with 1 mM salicylic acid at pH 7.0 enhanced the guaiacol peroxidase activity of the extracellular solution (so-called extracellular peroxidase). The soluble enzymes were partially purified by precipitation with ammonium sulfate followed by size exclusion and ion exchange chromatography. Despite an increase in the total activity of secreted peroxidase induced by pretreatment of excised roots with salicylic acid, the specific activity of the partially purified protein was significantly lower compared to that of the control. Purification of the corresponding proteins by ion exchange chromatography indicates that several isoforms of peroxidase occurred in both control and salicylic acid-treated samples. The activities of the extracellular peroxidases secreted by the salicylic acid-treated roots responded differently to calcium and lectins compared with those from untreated roots. Taken together, our data suggest that salicylic acid changes the isoforms of peroxidase secreted by wounded wheat roots. Received June 10, 2002; accepted September 24, 2002; published online May 21, 2003 RID="*" ID="*" Correspondence and reprints: Institute of Biochemistry and Biophysics, Russian Academy of Sciences, P.O. Box 30, Kazan 420111, Russia.     

Failure to corroborate claims that the developing endosperm of wheat (Triticum aestivum) contains significant activities of fructose-1,6-bisphosphatase

This work was done to test claims (Sangwan and Singh, Physiol. Plant. 73: 21–26) that the developing endosperm of wheat ( Triticum aestivum L.) contains a cytosolic and a plastidic fructose- 1,6-bisphosphatase (EC 3.1.3.11; FBPase). Repetition of the procedure of Sangwan and Singh with extracts of developing endosperm of Triticum aestivum cv. Mercia produced two peaks of apparent FBPase activity on elution from DEAE-cellulose. Both peaks showed high activity of pyrophosphate:fructose-6-phos-phate 1-phosphotransferase [EC 2.7.1.90; PFK(PP_i)]. The apparent FBPase activity in both peaks was stimulated by 20 μ M fructose-2,6-bisphosphate and inhibited by antibodies to PFK(PP_i). Antibody to plastidic FBPase did not react positively in an immunoblot analysis with any protein of M_r comparable to that of known FBPase in either peak. It is argued that the ability of each peak to convert fructose-1,6-bisphosphate to fructose-6-phosphate was due to PFK(PP_i). and that there remains no substantiated evidence for the presence of a plastidic FBPase in the developing endosperm of wheat.     

Salicylic acid and nitric oxide alleviate osmotic stress in wheat (Triticum aestivum L.) seedlings

Salicylic acid (SA) and nitric oxide (NO) are reported to alleviate the damaging effects of stress in plants rather similarly when applied at appropriate low concentrations. An experiment was therefore conducted to study the impact of SA, sodium nitroprusside (SNP; as NO donor), and methylene blue (MB; as a guanylate cyclase inhibitor) on wheat seedling performance under osmotic stress. Osmotic stress significantly reduced shoot fresh weight (SFW), chlorophyll contents (Chla, Chlb, total Chl), and membrane stability index (MSI) and also increased malondialdehyde (MDA) level, lipoxygenase (LOX) activity, and hydrogen peroxide production. Moreover, enzymatic antioxidant activities including superoxide dismutase, guaiacol peroxidase, and glutathione reductase activity were enhanced under osmotic stress. On the contrary, SA or SNP pretreatment reduced the damaging effects of osmotic stress by further enhancing the antioxidant activities that led to increased SFW, Chl, and MSI and reduced MDA level and LOX activity. However, pretreatment of plants with MB reversed or reduced the protective effects of SA and SNP suggesting that the protective effects were likely attributed to NO signaling. Therefore, NO may act as downstream of SA signaling in reduction of induced oxidative damage in wheat seedlings.     

Efficient callus induction and plant regeneration from mature embryo culture of winter wheat (Triticum aestivum L.) genotypes

Immature and mature embryos of 12 common winter wheat (Triticum aestivum) genotypes were cultured in vitro to develop an efficient method of callus formation and plant regeneration from mature embryo culture, and to compare the responses of both embryo cultures. Fifteen days after anthesis, immature embryos were aseptically dissected from seeds and placed with the scutellum upwards on a solid agar medium containing the inorganic components of Murashige and Skoog (MS) and 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). Mature embryos were moved slightly in the imbibed seeds. The seeds with moved embryos were placed furrow downwards in dishes containing 8 mg/l 2,4-D for callus induction. The developed calli and regenerated plants were maintained on 2,4-D-free MS medium. Plants regenerated from both embryo cultures were vernalized and grown to maturity in soil. Regenerated plantlets all maintained the hexaploid chromosome number. A strong genotypic effect on the culture responses was found for both explant cultures. Callus induction rate, regeneration capacity of callus and number of plants regenerated were independent of each other. Mature embryos had a high frequency of callus induction and regeneration capacity, and therefore, being available throughout the year, can be used as an effective explant source in wheat tissue culture. Received: 4 February 1997 / Revision received: 1 April 1997 / Accepted: 5 May 1997     

Prevalence and control of wheat (Triticum aestivum L.) seed borne fungi in farmer-saved seeds

High incidence of diseases associated with the use of seeds saved from previous harvests as desire of maintaining local varieties with special attributes is of increased concern in wheat industry worldwide. Prevalent of seed-borne fungi in farmer-saved seeds and seed dressing fungicides to prevent infection from seeds to seedlings was studied in Northern Tanzania. One hundred and thirty five untreated farmer-saved seed lots were collected randomly from farmers. Alternaria alternata, Bipolaris sorokiniana, Drechslera tritici, Fusarium graminearum, Fusarium moniliforme, Aspergillus flavus, Cladosporium sphaerospermum, Epicoccum purpurascens, Pyricularia oryzae and Penicillium corylophilum were fungi isolated in farmer-saved seeds. Mean seed infection was 29% causing average grain yield loss of 1.2 mt/ha^?1. Seed dressing with Metalaxy plus (Methyl carboxaitide), Mancozeb (Manganase-zinc salt) and Baytan (Chlorophenoxy ethanol) increased seed germination by 14, 13 and 17%, respectively, and grain yield by 28, 20 and 18%, respectively. Farmer-saved seeds were heavily infected by fungi with low grain yield performance.     

Amino acid metabolism associated with N-mobilization from the flag leaf of wheat (Triticum aestivum L.) during grain development

Field-grown winter wheat (Triticum aestivum L. cv. Castell) was used to study changes in the free amino acid pools of different plant parts and related enzyme activities in the flag leaf throughout the grain-filling period in three consecutive growing seasons. Amino acid analysis data indicated that, during senescence, the nitrogen flow in the flag leaf was directed towards the synthesis of glutamine as a specific nitrogen transport form. Of the enzymes involved, total glutamine synthetase (GS; EC 6.3.1.2) and especially ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1) activities declined continuously as senescence progressed. Unlike (chloroplastic) GS₂, (cytosolic) GS₁ was shown to be very persistent suggesting a special role for this isoenzyme in the N-reallocation process. Glutamate-oxaloacetate transaminase (GOT; EC 2.6.1.1), glutamate-pyruvate transaminase (GPT; EC 2.6.1.2) and isocitrate dehydrogenase (IDH; EC 1.1.1.42) showed a characteristic biphasic activity profile after anthesis. It is proposed that these enzymes, for each of which at least two isoenzymes were demonstrated, are involved in glutamate synthesis at the later stages of leaf senescence. Ammonium levels were fairly constant throughout the flag leafs life span, an ultimate rise often following peak values of glutamate dehydrogenase (GDH; EC 1.4.1.4) activity. The enzymology of flag leaf amino acid metabolism during grain development is further discussed in relation to observations of NH₃-volatilization from naturally senescing wheat plants.     

Identification of quantitative trait locus of zinc and phosphorus density in wheat (Triticum aestivum L.) grain

Zinc (Zn) is an essential micronutrient for human beings. However, Zn malnutrition has become a major problem throughout the world. Wheat is the most important food crop in the world, therefore, developing Zn-enriched wheat varieties provides an effective approach to reduce Zn malnutrition in human beings. The aim of this study was to understand the genetic control of grain Zn density in wheat and hence, to provide genetic basis for breeding wheat with high grain Zn density using molecular approach. A doubled haploid (DH) population developed from a cross between winter wheat varieties Hanxuan10 and Lumai 14 was used to identify quantitative trait loci (QTLs) for Zn concentration and content in wheat grains. In addition, phosphorus (P) concentration and content in wheat grain were also investigated to examine possible interactions between these two nutrients. The wheat grains used in this study were harvested from the plants grown under normal condition in a field trial. We found the grain Zn concentrations of the DH population varied from 25.9 to 50.5 mg/kg and the Zn content varied from 0.90 to 2.21 μg/seed. The grain P concentrations of the DH population varied from 0.258 to 0.429 mg/kg, and the P contents varied from 0.083 to 0.186 mg/seed. A significant positive correlation was observed between Zn and P density in this experiment. The results showed that both grain Zn and P densities were controlled by polygenes. Four and seven QTLs for Zn concentration and Zn content were detected, respectively. All the four QTLs for Zn concentration co-located with the QTLs for Zn content, suggesting a possibility to improve both grain Zn concentration and content simultaneously. Four and six QTLs for P concentration and P content were detected, respectively. The two QTLs for grain Zn concentration on chromosomes 4A and 4D co-located with the QTLs for P concentration. The four QTLs for grain Zn content on chromosome 2D, 3A and 4A co-located with the QTLs for P contents, reflecting the positive correlations between the grain Zn and P density, and providing possibility of improving grain micro- and macronutrient density simultaneously in wheat. In order to improve human health, the effect of P–Zn relation in grain on the Zn bioavailability should also be considered in the future work.     

Isolation and analysis of endophytic microorganisms in wheat (Triticum aestivum L.) leaves

The present investigation was undertaken in order to select the surface-sterilization technique most efficient for eliminating epiphytes, to document the spectrum of endophytes of healthy leaves from three wheat cultivars in Buenos Aires Province (Argentina) and to determine their infection frequencies at three growth stages. Surface-sterilization with undiluted commercial solution of sodium hypochlorite was reaffirmed as adequate for removing epiphytes on wheat leaves. From the 450 wheat leaf segments incubated, three bacterial isolates and 130 fungal isolates were obtained. From all the isolates, 19 fungal species were identified. Bacterial isolates were characterized as Bacillus sp. There were significant differences between microorganisms, stages of growth, and stages × microorganisms interaction. Differences between cultivars, stages × cultivars, microorganisms × cultivars and for the triple interaction were not significant. Frequency of microorganisms isolated increased with crop age, but it was statistically similar for the three wheat cultivars tested (Klein Centauro, Klein Dragón and Buck Ombú). Rhodotorula rubra, Alternaria alternata, Cladosporium herbarum and Epicoccum nigrum were isolated in the highest frequency. The other microorganisms were present at intermediate or low values. The species isolated may be assigned to three groups: (a) well-known and economically important pathogens of wheat, (b) commonly abundant phylloplane fungi considered to be primary saprobic and minor pathogens and (c) species occasionally present in wheat.     

Leaf primordia initiation,leaf emergence and tillering in wheat (Triticum aestivum L.) grown under low-phosphorus conditions

In two simultaneous experiments we examined the effects of phosphorus (P) supply on leaf area development in wheat (Triticum aestivum L.) grown in sand with nutrient solutions. In Experiment 1 we studied leaf emergence, leaf elongation, tiller emergence, shoot growth, and P uptake under four levels of P supply (mM) 0.025 (P1), 0.05 (P2), 0.1 (P3), and 0.5 (P4), and. In Experiment 2 there were two levels of P supply, P1 and P4, and we examined the effects of P on leaf primordia differentiation and leaf emergence. The phyllochron was calculated as the inverse of the rate of leaf emergence calculated from the regression of number of leaf tips (PHY-Ltip), Haun index (PHY-Haun), and as the cumulated thermal time between the emergence of two consecutive leaves (PHYtt). The plastochron was calculated from the inverse of the rate of leaf primordia initiation in the apex. P deficiency delayed the emergence of leaves on the main stem and on the tiller 1. Phosphorus deficiency increased the time from emergence to double ridge and anthesis. The final number of leaves was not affected by P. The effects of P on the value of the phyllochron were attributed to both a reduced rate of leaf primordia initiation, and to a reduced leaf elongation rate. P deficiency delayed or even suppressed the emergence of certain tillers. In this work a phosphorus deficiency that reduced shoot growth by 25% at 44 days after emergence significantly modified the structure of the plants by increasing the value of the phyllochron and delaying tillering. These results suggest that any attempt to simulate leaf area development and growth of wheat plants for P-limited conditions should include the effects of the deficiency on leaf emergence.     

The growth and phosphorus utilisation of plants in sterile media when supplied with inositol hexaphosphate, glucose 1-phosphate or inorganic phosphate

Seedlings of six temperate pasture species, three grasses and three legumes, were grown for 19–24 days in sterile agar or sand-vermiculite media, in the presence of inorganic phosphate (P_i), glucose 1-phosphate (G1P) or inositol hexaphosphate (IHP). Agar (pH 5.0) had a low IHP-sorbing capacity while IHP was almost completely sorbed in sand-vermiculite. P_i and G1P were relatively available in both media. Growth of each species was measured in relation to phosphorus (P) supply and levels of P_i supply at which shoot yields reached 90% of maximum yield (P_crit) were determined. P_crit values were generally higher for the legume species than for the grasses, and were six-fold higher for Trifolium subterraneum L. seedlings when grown in sand-vermiculite relative to agar. When supplied with G1P, seedlings of the six species grew as well as plants supplied with P_i. By contrast, IHP was a poor source of P for plant growth, even when supplied in agar at levels up to 40-fold greater than P_crit. Using the growth of T. subterraneum in the presence of IHP, it was calculated that roots released approximately 0.09 nkat phytase g^-1 root dry wt per day, over 20 days of growth. By supplementing agar containing IHP with phytase from Aspergillus niger (E.C. 3.1.3.8; 0.012 nkat plant^-1, or _∼1.3 nkat g^-1 root dry wt), sufficient P became available to enable T. subterraneum seedlings to grow as well as P_i-supplied plants. These results indicate that while pasture plants can quite effectively use P from some organic P sources (e.g. G1P), the acquisition of phytate-P is limited both by availability of substrate and the capacity of plant roots to hydrolyse available IHP. This revised version was published online in June 2006 with corrections to the Cover Date.     

Plant leaf area expansion and assimilate production in wheat (Triticum aestivum L.) growing under low phosphorus conditions

Under phosphorus deficiency reductions in plant leaf area have been attributed to both direct effects of P on the individual leaf expansion rate and to a reduced availability of assimilates for leaf growth. In this work we use experimental and simulation techniques to identify and quantify these processes in wheat plants growing under P-deficient conditions. In a glasshouse experiment we studied the effects of soil P addition (0–138 kg P₂O₅ ha^-1) on tillering, leaf emergence, leaf expansion, plant growth, and leaf photosynthesis of wheat plants (cv. INTA Oasis) that were not water stressed. Plants were grown in pots containing a P-deficient (3 mg P g^-1 soil) sandy soil. Sowing and pots were arranged to simulate a crop stand of 173 plants m^-2. Experimental results were integrated in a simulation model to study the relative importance of each process in determining the plant leaf area during vegetative stages of wheat. Phosphorus deficiency significantly reduced plant leaf area and dry weight production. Under P-deficient conditions the phyllochron (PHY) was increased up to a 32%, compared to that of high-P plants. In low-P plants the rate of individual leaf area expansion during the quasi-linear phase of leaf expansion (LER) was significantly reduced. The effect of P deficiency on LER was the main determinant of the final size of the individual leaves. In recently expanded leaves phosphorus deficiency reduced the photosynthesis rate per unit leaf area at high radiation (AMAX), up to 57%. Relative values of AMAX showed an hyperbolic relationship with leaf P% saturating at 0.27%. Relative values of the tillering rate showed an hyperbolic relationship with the shoot P% saturating at values above 0.38%. The value of LER was not related to the concentration of P in leaves or shoots. A morphogenetic model of leaf area development and growth was developed to quantify the effect of assimilate supply at canopy level on total leaf area expansion, and to study the sensitivity of different model variables to changes in model parameters. Simulation results indicated that under mild P stress conditions up to 80% of the observed reduction in plant leaf area was due to the effects of P deficiency on leaf emergence and tillering. Under extreme P-deficient conditions the simulation model failed to explain the experimental results indicating that other factors not taken into account by the model, i.e. direct effects of P on leaf expansion, must have been active. Possible mechanisms of action of the direct effects of P on individual leaf expansion are discussed in this work.