Isolation and comparative analysis of the wheat TaPT2 promoter: identification in silico of new putative regulatory motifs conserved between monocots and dicots.

Phosphorus deficiency is one of the major nutrient stresses affecting plant growth. Plants respond to phosphate (Pi) deficiency through multiple strategies, including the synthesis of high-affinity Pi transporters. In this study, the expression pattern of one putative wheat high-affinity phosphate transporter, TaPT2, was examined in roots and leaves under Pi-deficient conditions. TaPT2 transcript levels increased in roots of Pi-starved plants. A 579 bp fragment of the TaPT2 promoter is sufficient to drive the expression of the GUS reporter gene specifically in roots of Pi-deprived wheat. This TaPT2 promoter fragment was also able to drive expression of the GUS reporter gene in transgenic Arabidopsis thaliana, under similar growth conditions. Conserved regions and candidate regulatory motifs were detected by comparing this promoter with Pi transporter promoters from barley, rice, and Arabidopsis. Altogether, these results indicate that there are conserved cis-acting elements and trans-acting factors that enable the TaPT2 promoter to be regulated in a tissue-specific and Pi-dependent fashion in both monocots and dicots.

Low temperature responses of Nothofagus dombeyi and Nothofagus nitida, two evergreen species from south central Chile.

Nothofagus dombeyi (Mirb.) Blume and Nothofagus nitida (Phil.) Krasser are closely related evergreen trees native to south central Chile. Nothofagus dombeyi is a pioneer in habitats subject to high daytime irradiances and nighttime freezing temperatures and has a wider altitudinal and latitudinal distribution than N. nitida, which is restricted to more oceanic climates. We postulated that N. dombeyi has a greater cold-acclimation capacity, expressed as a greater capacity to maintain a functional photosynthetic apparatus at low temperatures, than N. nitida. Because cold-acclimation may be related to the accumulation of cryoprotective substances, we investigated relationships between ice nucleation temperature (IN), freezing temperature (FT), and the temperature causing injury to 50% of the leaf tissues (LT(50)) on the one hand, and concentrations of total soluble carbohydrates (TSC), starch and proline on the other hand. Observations were made throughout a seasonal cycle in adults and seedlings in the field and in seedlings in the laboratory under cold-acclimation inductive and non-inductive conditions. In adults, LT(50) values were lower in N. dombeyi than in N. nitida, suggesting that N. dombeyi is the more frost tolerant species. Adults of both species tolerated freezing in autumn and winter but not in spring and summer. In the fall and winter, seedlings of N. dombeyi had a much lower LT(50) than those of N. nitida. Nothofagus nitida seedlings, in autumn and winter, exhibited freezing avoidance mechanisms. Although elevated TSC and proline concentrations may contribute to freezing tolerance in adults of both species, an increase in proline concentration is unlikely to be the dominant frost tolerance response in adults because proline concentrations were higher in N. nitida than in N. dombeyi. In seedlings, however, there were large differences in proline accumulation between species that may account for the difference between them in freezing tolerance. Starch concentration in both species decreased during winter. Chlorophyll fluorescence indicated that maximal photochemical efficiency (F(v)/F(m)) remained at optimal values (~0.8) throughout the year. The effective photochemical efficiency of PSII (PhiPSII) and relative electron transport rates (ETR(r)) decreased in winter in both species. In seedlings, fluorescence parameters were more affected in winter in N. nitida than in N. dombeyi. We concluded that adults and seedlings of N. dombeyi are hardier than adults and seedlings of N. nitida, which is consistent with its wider latitudinal and altitudinal distribution.

Ethylene production and peroxidase activity in aphid-infested barley.

The purpose of this work was to investigate whether ethylene is involved in the oxidative and defensive responses of barley to the aphids Schizaphis graminum (biotype C) and Rhopalophum padi. The effect of aphid infestation on ethylene production was measured in two barley cultivars (Frontera and Aramir) that differ in their susceptibility to aphids. Ethylene evolution was higher in plants infested for 16 hr than in plants infested for 4 hr in both cultivars. Under aphid infestation, the production of ethylene was higher in cv. Frontera than in Aramir, the more aphid susceptible cultivar. Ethylene production also increases with the degree of infestation. Maximum ethylene evolution was detected after 16 hr when plants were infested with 10 or more aphids. Comparing the two species of aphids, Schizaphis graminum induced more ethylene evolution than Rhopalosiphum padi. Infestation with S. graminum increased hydrogen peroxide content and total soluble peroxidase activity in cv. Frontera, with a maximum level of H2O2 observed after 20 min of infestation and the maximum in soluble peroxidase activity after 30 min of infestation. When noninfested barley seedlings from cv. Frontera were exposed to ethylene, an increase in hydrogen peroxide and in total peroxidase activity was detected at levels similar to those of infested plants from cv. Frontera. When noninfested plants were treated with 40 ppm of ethylene, the maximum levels of H2O2 and soluble peroxidase activity were at 10 and 40 min, respectively. Ethylene also increased the activity of both cell-wall-bound peroxidases types (ionically and covalently bound), comparable with infestation. These results suggest that ethylene is involved in the oxidative responses of barley plants induced by infestation.

Induction of soluble and cell wall peroxidases by aphid infestation in barley.

Peroxidase enzymes have been found in soluble, ionically bound, and covalently bound forms and have been implicated in several physiological processes in plants. This paper investigates the effect of aphid infestation on soluble and bound-cell wall peroxidase activity and bound-cell wall isoform changes of barley plants. Peroxidase activity was measured in control plants and plants infested with the aphid Schizaphis graminum (Rondani). The activity of soluble peroxidases increased with time of infestation, older plants being more affected than younger ones. The increase in bound-cell wall peroxidase activity as a function of age was higher in infested than in control plants, being higher in ionically bound than in covalently bound peroxidases. When the aphids were removed from plants, the activities of both types of peroxidases decreased to control levels. Isoelectrofocusing analyses of the ionically bound peroxidases showed changes in the isoform pattern. A new isoform was induced by infestation. The activities of all covalently bound isoforms increased after infestation. The physiological implications of these changes are discussed.

Plant proteinase inhibitors: a defensive response against insects. (Review).

Plants protect themselves against pests using their wide chemical defense arsenal. Among several defense proteins, proteinase inhibitors appear to be an important group. Proteinase inhibitors are widely present in plants and they are often found in storage organs. They are known to be inducible in plants by injuries, such as insect damage. Because these proteins inhibit digestive enzymes of insect larvae and microbial proteases, they may be considered as mechanisms to improve the plant defense against pests. In recent years, growing research on plant proteinase inhibitors has confirmed their important role in plant defense, although several aspects are still controversial. Although many plants have related proteinase inhibitors, which have been shown to affect metabolism and/or development of different insects, these plants do not seem to share a common inhibitor induction mechanism. This is an emerging field and much work is yet to be done.

Plant proteinase inhibitors: a defensive response against insects. (Review)

Plants protect themselves against pests using their wide chemical defense arsenal. Among several defense proteins, proteinase inhibitors appear to be an important group. Proteinase inhibitors are widely present in plants and they are often found in storage organs. They are known to be inducible in plants by injuries, such as insect damage. Because these proteins inhibit digestive enzymes of insect larvae and microbial proteases, they may be considered as mechanisms to improve the plant defense against pests. In recent years, growing research on plant proteinase inhibitors has confirmed their important role in plant defense, although several aspects are still controversial. Although many plants have related proteinase inhibitors, which have been shown to affect metabolism and/or development of different insects, these plants do not seem to share a common inhibitor induction mechanism. This is an emerging field and much work is yet to be done

Effect of ursolic acid from epicuticular waxes of Jacaranda decurrens on Schizaphis graminum.

Ursolic acid from Jacaranda decurrens showed toxicity and feeding deterrency towards the greenbug Schizaphis graminum. Biological activity was determined by analyzing ursolic acid effects on the survival, reproductive index, and population growth rate of the greenbug. Survival and reproductive index decreased in direct proportion to ursolic acid content in the diet. The population growth rate decreased markedly when the aphids were fed on barley leaves sprayed with ursolic acid dissolved in DMSO, in comparison to leaves sprayed only with DMSO. The feeding behavior of the greenbug was also affected by ursolic acid. Ingestion time on diet with 0.1 mM was reduced about 30% in relation to the ingestion time on control diet.

Biosynthesis of Indol-3-yl-acetyl-myo-inositol Arabinoside in Kernels of Zea mays L.

Extracts of immature kernels of Zea mays L. catalyzed the synthesis of indol-3-yl-acetyl-myo-inositol arabinoside from indol-3-yl-acetyl-myo-inositol and UDP-[U-(14)C]xylose. The product contained radioactivity which upon hydrolysis with trifluoroacetic acid cochromatographed with arabinose and not xylose. The amount of product from the reaction was proportional to the amount of indol-3-yl-acetyl-myo-inositol added, and the product was positive to Ehmann's reagent for indoles. In addition, the product and authentic indol-3-yl-acetyl-myo-inositol arabinoside had the same R(F) or retention time in three chromatographic systems.By analogy to the wheat germ system, it is proposed that a UDP-d-xylose 4-epimerase is present in immature kernels of maize and that this enzyme catalyzes the conversion of UDP-d-xylose to UDP-l-arabinose, the probable sugar donor to indol-3-yl-acetyl-myo-inositol.The present data demonstrates the presence of a previously undescribed enzyme, UDP-arabinose:indol-3-yl-acetyl-myo-inositol arabinosyl transferase in maize kernels. This work together with our prior studies provides a pathway for the biosynthesis of all the low molecular weight esters of indol-3-yl-acetic acid in maize kernels.

Enzymic synthesis of indol-3-ylacetyl-myo-inositol galactoside.

Extracts of immature kernels of Zea mays catalysed the synthesis of indol-3-ylacetyl-myo-inositol galactoside from indol-3-ylacetyl-myo-inositol and UDP-galactose. Addition of 2-mercaptoethanol was required for stability of the catalytic activity during dialysis. The enzyme could be fractionated with (NH4)2SO4, and 55% of the activity was recovered in the 30-60%-saturation fraction. The product of the reaction contained radioactivity from UDP-[U-14C]galactose and was identified as indol-3-ylacetyl-myo-inositol galactoside by gas chromatography-mass spectrometry. Therefore a UDP-galactose:indol-3-ylacetyl-myo-inositol galactosyltransferase (indol-3-ylacetyl-myo-inositol galactoside synthase) is present in developing kernels of Zea mays. The description of this enzyme, together with the enzymes described in the accompanying paper [Michalczuk & Bandurski (1982) Biochem. J. 207, 273-281] for the synthesis of indol-3-ylacetylglucose and indol-3-ylacetyl-myo-inositol, now provides mechanisms for the biosynthesis of one-half of the low-molecular-weight esters of indol-3-ylacetic acid in Zea mays.

Effects of Hydroxamic Acids Isolated from Gramineae on Adenosine 5'-triphosphate Synthesis in Chloroplasts.

Two hydroxamic acids isolated from maize extracts, 2,4-dihydroxy-7-methoxy-1,4-(2H)-benzoxazin-3(4H)-one (DIMBOA) and the 2-O-beta-d-glucopyranoside of DIMBOA, inhibit photophosphorylation by spinach chloroplasts. Both cyclic and noncyclic photophosphorylations were inhibited to the same extent. The concentrations producing 50% inhibition for DIMBOA and its glucoside were about 1 and 4 millimolar, respectively. These compounds inhibit coupled electron transport but do not affect basal or uncoupled electron transport. Both acids inhibit the ATPase activities of membrane-bound coupling factor 1 (CF(1)) and of purified CF(1). On the basis of these results, it is concluded that DIMBOA and its glucoside act as energy transfer inhibitors of photophosphorylation.

Quantitation of 1,4-Benzoxazin-3-ones in Maize by Gas-Liquid Chromatography.

A gas-liquid chromatographic (GLC) procedure is reported for the quantitation of the trimethylsilyl (TMS) derivatives of substituted 2-hydroxy-2H-1,4-benzoxazin-3(4H)-ones (2-hydroxy-2H-1,4-benzoxazin-3(4H)-one[HBOA]; 2-hydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one[HMBOA];2,4- dihydroxy-2H-1,4-benzoxazin-3(4H)-one[DIBOA]; 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one[DIMBOA]; and 2,4-dihydroxy-7,8-dimethoxy-2H-1,4-benzoxazin-3(4H)-one[DIM (2)BOA]) found in maize (Zea mays L.) extracts. Derivatized samples were chromatographed on columns with liquid phases of 2% DC-11 and 3% OV-17 and detected by flame ionization. Internal standards were methyl palmitate and methyl stearate on DC-11 and methyl behenate on OV-17. Detector response was linear to at least 5 nanomoles for TMS(2)-HBOA and TMS(2)-DIBOA and to 19 nanomoles for TMS(2)-DIMBOA. Standard errors of 2% or less were obtained when four replicate samples were analyzed. For each of the 15 maize lines examined, the amount of DIMBOA determined by GLC was directly proportional to the amount of ferric chloride-reactive material determined colorimetrically.

Identification of 1,4-Benzoxazin-3-ones in Maize Extracts by Gas-Liquid Chromatography and Mass Spectrometry.

Gas-liquid chromatography-mass spectrometry (GLC-MS) has been used for the separation, detection, and identification of 1,4-benzoxazin-3-ones (hydroxamic acids and lactams) and benzoxazolinones found in maize (Zea mays L.) extracts. Compounds of interest were partitioned into ethyl acetate from aqueous maize seedling extracts. For analysis by GLC-MS, trimethylsilyl derivatives were prepared, chromatographed on a column of 3% OV-1, and detected in the mass spectrometer. Mass spectra were obtained for all peaks present in extracts of four maize lines. A data comparison system was developed for relating unidentified spectra to the spectra of the reference compounds. Based on spectral comparisons, three hydroxamic acids (2,4-dihydroxy-2H-1, 4-benzoxazin-3(4H)-one; 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one; and 2,4-dihydroxy-7,8-dimethoxy-2H-1,4-benzoxazin-3(4H)-one), three lactams (2-hydroxy-2H-1,4-benzoxazin-3(4H)-one; 2,7-dihydroxy-2H-1,4-benzoxazin-3(4H)-one; and 2-hydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one), one benzoxazolinone (6-methoxybenzoxazolinone), and two organic acids (malic and aconitic) were identified in the extracts. In addition, one other hydroxamic acid and one other related compound were tentatively identified based on mass spectral evidence.

2,4-Dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one, an Inhibitor from Zea mays with Differential Activity against Soft Rotting Erwinia Species.

[2,4-Dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one] DIMBOA was extracted with ethyl acetate from acidified water homogenates of corn (Zea mays L.) seedlings. Pure DIMBOA or ethyl acetate extracts of corn tissue were added to bacterial growth medium at five concentrations (measured as hydroxamates). DIMBOA and corn extracts were more inhibitory to soft rot bacteria (Erwinia spp.) that are nonpathogenic to corn than to soft rot bacteria that are corn pathogens. The inhibitory activity of DIMBOA was similar to that of the ethyl acetate extracts. Both corn extracts and DIMBOA prolonged the lag phase of bacterial growth without significantly changing log phase growth rates. At various concentrations of the inhibitor, 50 to 100% of the activity of corn extracts inhibitory to different bacterial isolates was attributable to DIMBOA. Extracts of DIMBOA-deficient plants (genotype bxbx) were not inhibitory to Erwinia spp. It was concluded that DIMBOA is the major active component in those corn extracts which are inhibitory to soft rot Erwinia species.

Decomposition of 2,4-Dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one in Aqueous Solutions.

Cyclic hydroxamic acids present in some species of Gramineae have been reported to be important in resistance of these plants to fungi and insects. Since the nonglucosylated forms of these acids are unstable in aqueous solution, in vitro methods for the measurement of their antibiotic properties have been difficult. Kinetics of the decomposition of 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA), the major hydroxamate in corn (Zea mays L.) extracts, were studied in buffered aqueous solutions from pH 5 to 7.5 at temperatures from 20 to 80 C. Kinetics were apparently first order under all conditions tested; energies of activation (24 to 28 kcal/mol) were nearly pH-independent. DIMBOA decomposed rapidly (half-life = 5.3 hours at 28 C, pH 6.75) relative to the time required for many procedures which have been used to demonstrate the biological activity of DIMBOA. The rate of disappearance of inhibitory activity of DIMBOA toward Erwinia carotovora was indistinguishable from the rate of decomposition of DIMBOA. Contrary to reports, yields of 6-methoxy-2-benzoxazolinone (MBOA) were not quantitative. Gas-liquid chromatography analytical procedures were developed for quantitation of trimethylsilyl and acetyl derivatives of MBOA. As measured by ultraviolet spectroscopy and/or gas-liquid chromatography, conversion of DIMBOA to MBOA ranged from 40 to 75% of theoretical in aqueous buffers, bacterial growth medium, and ethyl acetate extracts of corn tissue resuspended in buffer. Yields varied with temperature, pH, and constituents in the medium.

Factors That Influence the Activity of 2,4-Dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one on Erwinia Species in Growth Assays.

Factors affecting the inhibitory activity of 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA) against Erwinia carotovora, a nonpathogen of Zea mays L., and against a maize pathovar of Erwinia chrysanthemi (ECZ) were examined. Most experiments were performed with DIMBOA dissolved in a bacterial growth medium containing 10 g/liter of sucrose, inorganic salts, and 1 g/liter of casamino acids at pH 6.75. When temperature and pH were held constant, inhibition of E. carotovora varied linearly with the logarithm of the initial cell population. By altering temperatures, assays with constant pH and initial cell populations were performed under conditions of varying DIMBOA stability. When E. carotovora was grown at 24, 28, 32, and 36 C in the presence of 0.1 to 0.5 mm DIMBOA, the inhibition of bacterial growth was maintained long after DIMBOA had decomposed in the medium to levels which, if added initially, would not have been inhibitory. When assays were performed at pH 5.5, the pH of aqueous maize extracts, E. carotovora was more inhibited than at pH 6.75; however, ECZ was substantially less inhibited at the lower pH.