Purification and Characterization of an Iminopeptidase from the Primary Leaf of Wheat (Triticum aestivum L.)

Iminopeptidase (EC 3.4.11.5) was substantially purified from the primary leaves of 7-day-old wheat seedlings (Triticum aestivum L.). The purification procedure consisted of five steps: acid precipitation, molecular exclusion chromatography on Sephacryl S-200, Ultrogel AcA 44, Sepharose 2B and ion-exchange chromatography on DEAE-cellulose. Iminopeptidase isolated in this manner was only active against the β-naphthylamides of proline and hydroxyproline. For each substrate, the pH optimum was 7.4 and activity was sensitive to sulfhydryl group inhibitors. The iminopeptidase hydrolyzed the dipeptides Pro-Leu, Pro-Gly, Hyp-Gly, and Pro-Tyr. Iminopeptidase activity against the dipeptide Pro-Gly was higher than against Hyp-Gly. The molecular weight was estimated to be about 400,000. Evidence was obtained for the existence of endogenous inhibitors of iminopeptidase activity.     

Isolation and Some Properties of an Aminopeptidase from the Primary Leaf of Wheat (Triticum aestivum L.)

Evolution of nitrogen oxides (NO_(x), primarily as nitric oxide) from soybean (Glycine max [L.] Merr.) leaves during purged in vivo nitrate reductase assays had been reported; however, these reports were based on a method that had been used for determination of NO_(x) in air. This method also detects other N compounds. Preliminary work led us to doubt that the evolved N was nitric oxide. Studies were undertaken to identify the N compound evolved from the in vivo assay that had been reported as NO_(x). Material for identification was obtained by cryogenic trapping and fractional distillation, and by chemical trapping procedures. Mass spectrometry, ultraviolet spectroscopy, and ¹⁵N-labeled nitrate were used to identify the compounds evolved and to determine whether these compounds were derived from nitrate. Acetaldehyde oxime was identified as the predominant N compound evolved and this compound is readily detected by the method for NO_(x) determination. Substantial quantities of acetaldehyde oxime (16.2 micromoles per gram fresh weight per hour) were evolved during the in vivo assay. Small amounts of nitrous oxide (0.63 micrograms N per gram fresh weight per hour) were evolved, but this compound is not detected as NO_(x). Acetaldehyde oxime and nitrous oxide were both produced as a result of nitrate (¹⁵NO₃⁻) reduction during the assay.     

In Vitro Stability of Nitrate Reductase from Wheat Leaves: II. Isolation of Factors from Crude Extract Which Affect Stability of Highly Purified Nitrate Reductase

When a crude extract from 8-day-old wheat (Triticum aestivum L. cv. Olympic) leaves was fractionated by a combination of ammonium sulfate precipitation and Sephadex G-100 chromatography the presence of three factors which have a marked effect on the stability of highly purified nitrate reductase was revealed. Two of these factors (I and III) have a positive effect and the other factor (II) has a negative effect on stability. Factors I and III can each overcome the instability-promoting effect of II; however, this was apparently not due to a direct effect on factor II.     

Proteolytic enzymes in green wheat leaves I. Isolation on DEAE-cellulose of several proteinases with acid pH optima

Several peptide hydrolases have been isolated in a partiallypurified form from green wheat leaves by a combination of gelchromatography and chromatography on DEAE-cellulose. These hydrolasesall degrade haemoglobin and other substrates tested at an acidpH. Three of the enzymes have been identified as proteinasesby measurement of the total N: -amino N ratio of the degradationproducts. These three enzymes have a similar optimum assay pHof about 4.5 with haemoglobin as substrate, but differ markedlyin their stability at high temperatures. ¹ Nomenclature in this paper follows the recommendation of theCommission on Biochemical Nomenclature (1972). (Received November 30, 1977; )     

Degradation of ribulose-1,5-bisphosphate carboxylase by proteolytic enzymes from crude extracts of wheat leaves

In crude extracts from the primary leaf of wheat seedlings, Triticum aestivum L., cv. Olympic, maximum proteinase activity, as determined by measuring the rate of release of amino nitrogen from ribulose-bisphosphate carboxylase (RuBPCase), was found to be obtained only when EDTA and L-cysteine were included in the extraction buffer. Highest proteinase activity was obtained by grinding at pH 6.8, although the level of activity was similar in the pH range 5.6 to 8.0; this range also coincided with maximum extractability of protein. The lower amount of RuBPCase degrading proteinase extracted at low pH was not due to an effect of pH on enzyme stability. The optimum temperature of reaction was 50° C and reaction rates were linear for at least 120 min at this temperature. In the absence of substrate the proteinase was found to be very sensitive to temperatures above 30° C, with even short exposures causing rapid loss of activity. The relation between assay pH and RuBPCase degradation indicated that degradation was restricted to the acid proteinase group of enzymes, with a pH optimum of 4.8, and no detectable activity at a pH greater than 6.4. The levels of extractable RuBPCase proteinase exhibited a distinct diurnal variation, with activity increasing during the latter part of the light period and then declining once the lights were turned off. The effect of leaf age on the level of RuBPCase, RuBPCase proteinase and total soluble protein was investigated. Maximum RuBPCase activity occurred 9 days after sowing as did soluble protein. After the maximum level was obtained, the pattern of total soluble protein was shown to be characterised by three distinct periods of protein loss: I (day 9–13) 125 ng leaf^-1 day^-1; II (day 15–27) 11 ng leaf^-1 day^-1; III (day 29–49) 22 ng leaf^-1 day^-1. Comparison of the pattern of RuBPCase activity and total protein suggest that the loss of RuBPCase may be largely responsible for the high rate of protein loss during period I. Proteinase activity increased sharply during the period of most rapid loss of RuBPCase activity, and because the specific activity of RuBPCase also declined, we concluded that RuBPCase was being degraded more rapidly than the other proteins. Once the majority of the RuBPCase was lost, there did not appear to be a direct relation between RuBPCase proteinase activity and rate of total soluble protein loss, since the proteinase exhibited maximum activity during the slowest period of protein loss (II), and was declining in activity while the rate of protein loss remained stable during the third and final period of total protein loss.Abbreviations RuBPCase ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) - TCA trichloroacetic acid Supported by the Wheat Industry Research Council of Australia and the Australian Research Grants Committee D2 74/15052     

Nitrogen redistribution during grain growth in wheat (Triticum aestivum L.)

The technique of EDTA-enhanced phloem exudation (King and Zeevaart, 1974: Plant Physiol. 53, 96–103) was evaluated with respect to the collection and identification of amino acids exported from senescing wheat leaves. Whilst the characteristics of the exudate collected conform with many of the accepted properties of phloem exudate, unexpectedly high molar proportions of phenylalanine and tyrosine were observed. By comparing exudation into a range chelator solutions with exudation into water, the increased exudation of phenylalanine and tyrosine relative to the other amino acids occurring when ethylene-diaminetetracetic acid was used, was considered to an artefact.In plants thought to be relying heavily on mobilisation of protein reserves to satisfy the nitrogen requirements of the grain, the major amino acids present in flag-leaf phloem exudate were glutamate, aspartate, serine, alanine and glycine. Only small proportions of amides were present until late in senescence when glutamine became the major amino acid in phloem exudate (25 molar-%). Glutamine was always the major amino acid in xylem sap (50 molar-%).The activities of glutamine synthetase (EC 6.3.1.2), glutamate synthase (EC 1.4.7.1), glutamate dehydrogenase (EC 1.4.1.3) and asparagine synthetase (EC 5.3.5.4) were measured in the flag leaf throughout the grain-filling period. Glutamine synthetase and glutamate-synthase activities declined during this period. Glutamate-dehydrogenase activity was markedly unchanged despite variation in the number of multiple forms visualised after gel electrophoresis. The activity of the enzyme reached a peak only very late in the course of senescence of the flag leaf. No asparagine-synthetase activity could be detected in the flag leaf during the grain-filling period.II. Peoples et al. (1980)     

Phylogenetic Framework for Trema (Celtidaceae)

We used ITS and trnL sequence data, analyzed separately and combined by MP, to explore species relationships and concepts in Trema (Celtidaceae), a pantropical genus of pioneer trees. Whether Trema is monophyletic or includes Parasponia is still unresolved. Three clades within Trema received moderate to high support, one from the New World and two from the Old World, but their relationships were not resolved. In the New World, specimens of T. micrantha formed two groups consistent with endocarp morphology. Group I, with smaller brown endocarps, is a highly supported clade sister to T. lamarckiana. Group II, with larger black endocarps, is poorly resolved with several subclades, including the highly supported T. integerrima clade. Both Old World clades contain Asian and African species, with three or more species in each region. Trema orientalis is not monophyletic: specimens from Africa formed a highly supported clade sister to T. africana, while those from Asia were sister to T. aspera from Australia.     

An ectomycorrhizal nitrogen economy facilitates monodominance in a neotropical forest

Tropical forests are renowned for their high diversity, yet in many sites a single tree species accounts for the majority of the individuals in a stand. An explanation for these monodominant forests remains elusive, but may be linked to mycorrhizal symbioses. We tested three hypotheses by which ectomycorrhizas might facilitate the dominance of the tree, Oreomunnea mexicana, in montane tropical forest in Panama. We tested whether access to ectomycorrhizal networks improved growth and survival of seedlings, evaluated whether ectomycorrhizal fungi promote seedling growth via positive plant–soil feedback, and measured whether Oreomunnea reduced inorganic nitrogen availability. We found no evidence that Oreomunnea benefits from ectomycorrhizal networks or plant–soil feedback. However, we found three‐fold higher soil nitrate and ammonium concentrations outside than inside Oreomunnea‐dominated forest and a correlation between soil nitrate and Oreomunnea abundance in plots. Ectomycorrhizal effects on nitrogen cycling might therefore provide an explanation for the monodominance of ectomycorrhizal tree species worldwide.     

Topographic position affects the water regime in a semideciduous tropical forest in Panamá

The effects of topographic position on water regime in a semideciduous tropical forest on Barro Colorado Island in Panamá were assessed by measuring soil matric potential using the filter paper technique and by using measured soil water release characteristics to convert a long-term (20 years) gravimetric water content data-set to matric potential. These were also compared against predictions from a simple water balance model. Soil matric potentials on slope sites were significantly higher than on plateau sites throughout the measurement interval and slopes experienced a shorter duration of drought during the annual dry-season. Measured values of matric potential agreed with those predicted from converting the gravimetric measurements using water release characteristics. Annual duration of drought predicted by the simple water balance model agreed with values determined from the converted long term water content data-set and was able to predict the annual duration of drought on plateau sites. On slope sites, the water balance systematically and significantly overestimated the duration of drought obtained from the water content data-set, suggesting that slope sites were supplied with water from upslope. Predictions of annual drought duration from sites with higher annual rainfall than Barro Colorado Island (BCI), suggest that while plateau sites on BCI experience a water regime consistent with annual rainfall, slopes experience a water regime more similar to that of forests with much higher rainfall. We conclude that such large variations in water regime over small spatial scales may play a role in maintaining high species richness through providing opportunities for niche specialisation and by buffering slopes against possible climate change.