Ineffectiveness of Abscisic Acid in Stomatal Closure of Yellow Lupin, Lupinus luteus var. Weiko III

Stomata of yellow lupin leaves are remarkably insensitive toabscisic acid (ABA). Stomatal resistance was monitored usingboth a viscous now porometer and a diffusion porometer. Resultswere confirmed with scanning electron microscopy. When exogenousABA solutions were supplied via petioles, 10^–6 M solutionshad no effect on stomatal resistance. Upper (adaxial) stomatawere not affected by 10^–5 M ABA but lower stomata showed3-fold more resistance after 2 h. Stomata of both surfaces closedafter 30 min in 10^–4 M ABA. Isolated epidermal peels of lupin leaves were floated on ABAsolutions yet upper surface peels showed no stomatal closingin 10^–4 M ABA, while lower surface stomata closed to abarely significant extent. Stomata of intact leaves were not very sensitive to darkness,showing at most a doubling in resistance after 6 h darkness.Complete stomatal closure, however, was readily produced bywilting leaves. Hence, lupin stomata are physically capableof closing. Endogenous ABA levels of water-stressed leaves increased approximately10-fold, which corresponds to concentrations below 10 µMABA. It is concluded that ABA is unlikely to play a role incontrolling short-term stomatal response of lupins.     

The Role of Abscisic Acid in Flower Abscission of Lupinus luteus

The role of abscisic acid in the control of flower abscission in Lupinus luteus L. was examined. Using a modified extraction and purification technique, endogenous abscisic acid levels in the upper flowers of an inflorescence were found to increase markedly some days before abscission could be detected. When abscisic acid was injected into flower-bearing nodes or fed via the roots, no increase in the abscission rate was obtained at any position in the flowerhead. Application of abscisic acid to only the leaves resulted in a marked increase in flower abscission. The role of abscisic acid per se as a primary controlling factor of flower abscission in yellow lupin is questioned.     

Some Structural Changes During Ripening of Mangoes (Mangifera indica var. Alphonso) by Abscisic Acid Treatment

Abscisic acid at 10^–6 M concentration enhances ripeningof mangoes. The cells in the pulp of the fruit are large andparenchymatous and lose their integrity due to cell wall hydrolysisat the ripe stage. They contain abundant polysaccharides, consistingmainly of starch, which is degraded during ripening. The chloroplastsare transformed to chromoplasts containing red or yellow carotenoidpigment. Abscisic acid treatment enhances all of these processes.Mitochondria, on the other hand, retain their structural integritythroughout the ripening process in untreated and abscisic acid-treatedmangoes. Mangoes, ripening, abscisic acid, structure     

Yellow lupin (Lupinus luteus) aminoacyl-tRNA synthetases. Isolation and some properties of enzyme-bound valyl adenylate and seryl adenylate.

As a continuation of our studies on plant (yellow lupin, Lupinus luteus) aminoacyl-tRNA synthetases we describe here formation and some properties of valyl-tRNA synthetase-bound valyl adenylate (EVal(Val-AMP)) and seryl-tRNA synthetase-bound seryl adenylate (ESer(Ser-AMP)). Valyl-tRNA synthetase-bound valyl adenylate was detected and isolated by several approaches in the pH range 6--10. In that range inorganic pyrophosphatase increases the amount of valyl adenylate by factor 1.8 regardless of pH. 50% of valine from the EVal(Val-AMP) complex isolated by Sephadex G-100 gel filtration was transferred to tRNA with a rate constant greater than 4 min-1 (pH 6.2, 10 degrees C). The ratio of valine to AMP in the enzyme-bound valyl adenylate is 1 : 1 and it is not changed by the presence of periodate-oxidized tRNA. In contrast to enzyme-bound valyl adenylate, formation of ESer(Ser-AMP) is very sensitive to pH. Inorganic pyrophosphatase increases the amount of seryl adenylate by a factor 6 at pH 8.0 and 30 at pH 6.9 60% of serine from the ESer(Ser-AMP) complex was transferred to tRNA with a rate constant greater than 4 min-1 (pH 8.0, 0 degrees C). The ratio of serine to AMP in the enzyme-bound seryl adenylate is 1 : 1. The rate of synthesis of the enzyme-bound aminoacyl adenylates was measured by ATP-PPi exchange. Michaelis constants for the substrates of valyl-tRNA and seryl-tRNA synthetases in ATP-PPi exchange were determined. Effects of pH, MgCl2 and KCl on the initial velocity of aminoacyl adenylate formation are described. For comparison, catalytic indices in the aminoacylation reactions catalyzed by both lupin enzymes are given and effects of pH, MgCl2 and KCl on tRNA aminoacylation are presented as well. Under some conditions, e.g. at low pH or high salt concentration, lupin valyl-tRNA and seryl-tRNA synthetase are active exclusively in ATP-PPi exchange reaction.     

Aminoacylation of four tRNA species in lupin (Lupinus luteus) cotyledons

During germination of lupin seeds, the levels of in-vivo tRNA aminoacylation increase in different ways, depending on the species of tRNA. Column chromatography of tRNA on reverse-phase-chromatography (RPC-5) has shown the presence of 4 peaks of isoleucyl-tRNA, 5 of leucyl-tRNA, 5 of lysyl-tRNA, 2 of tyrosyl-tRNA, and 4 of valyl-tRNA. Cochromatography of periodate treated and control tRNA preparations, labeled with radioactive amino acids, indicates identical aminoacylation in vivo of isoaccepting tRNAs during plant development. One isoacceptor of isoleucine tRNA changes its elution profile after periodate treatment.Abbreviation RPC-5 reverse-phase-chromatography     

Identification of a Low Digestibility δ-Conglutin in Yellow Lupin (Lupinus luteus L.) Seed Meal for Atlantic Salmon (Salmo salar L.) by Coupling 2D-PAGE and Mass Spectrometry

The need of quality protein in the aquaculture sector has forced the incorporation of alternative plant proteins into feeding diets. However, most plant proteins show lower digestibility levels than fish meal proteins, especially in carnivorous fishes. Manipulation of protein content by plant breeding can improve the digestibility rate of plant proteins in fish, but the identification of low digestibility proteins is essential. A reduction of low digestibility proteins will not only increase feed efficiency, but also reduce water pollution. Little is known about specific digestible protein profiles and/or molecular identification of more bioavailable plant proteins in fish diets. In this study, we identified low digestibility L. luteus seed proteins using Atlantic salmon (Salmo salar) crude digestive enzymes in an in vitro assay. Low digestibility proteins were identified by comparing SDS-PAGE banding profiles of digested and non-digested lupin seed proteins. Gel image analysis detected a major 12 kDa protein band in both lupin meal and protein isolate digested products. The 12 kDa was confirmed by 2D-PAGE gels and the extracted protein was analyzed with an ion trap mass spectrometer in tandem mass mode. The MS/MS data showed that the 12 kDa low digestibility protein was a large chain δconglutin, a common seed storage protein of yellow lupin. Comparison of the protein band profiles between lupin meal and protein isolates showed that the isolatation process did not affect the low digestibility of the 12 kDa protein.     

Diurnal Changes in Endogenous Abscisic Acid in Leaves of Pearl Millet (Pennisetum americanum (L. ) Leeke) under Field Conditions

Diurnal variation in leaf abscisic acid (ABA) content was investigatedin pearl millet (Pennisetum americanum (L. ) Leeke) growingin the field in the semi-arid tropics and subjected to varyingdegrees of water stress. There was a two- to three-fold change in ABA content duringthe photoperiod in three groups of ‘severely’ stressedplants of the genotype BJ 104. Maximum ABA occurred mid-morning(1030 h). ABA levels then declined to a minimum at 1500 h. Changesin ABA content of ‘moderately’ stressed and fullyirrigated plants were smaller, but still significant. Though,when averaged over the day, levels of ABA of the five groupswere positively related to the degree of water stress, relationshipsbetween ABA concentration and total water () or turgor (_p) potentialsvaried considerably with time of sampling. Within groups, changesin ABA contents during the day were not always accounted forby changes in or _p. Temporal changes in leaf ABA content similar to those foundin BJ 104, and largely unrelated to , were observed in the genotypesSerere 39 and B282 in a subsequent year. Leaf ABA content of droughted plants (BJ 104) did not declineappreciably overnight despite a marked increase in . However,a large reduction in ABA content with increase in did occurfollowing heavy rainfall. Diurnal changes in stomatal conductance (g₁) of BJ 104 couldnot be simply accounted for by temporal changes in total leafABA content, even when allowance was made for effects of irradianceand other environmental variables on g₁. It is suggested thatthe sensitivity of stomata to ABA, or accessibility of the hormoneto the stomatal complex, changes during the day.     

Aphids do not avoid resistance in Australian lupin (Lupinus angustifolius, L. luteus) varieties

Laboratory bioassays and field trials were used to characterize resistance to three aphid species (Myzus persicae (Sulzer), Acyrthosiphon kondoi Shinji, Aphis craccivora (Koch) in two aphid-resistant varieties (Kalya, Tanjil) and one susceptible variety (Tallerack) of Lupinus angustifolius L., and in one resistant variety (Teo) and one susceptible variety (Wodjil) of L. luteus L. Host selection tests in the glasshouse showed that alates of all three species preferred L. luteus to L. angustifolius, but provided no evidence that alates selected susceptible varieties over resistant. These results were supported by a field trial, which showed no difference in the number of colonizing A. kondoi alates collected from the resistant and susceptible lines of each lupin species, but there were significantly more late-instar nymphs and apterous adults on the susceptible lines. In laboratory host suitability experiments, there was much greater suppression of aphid growth and survival on Teo than on Kalya and Tanjil. In field trials, the numbers of aphids were generally lower on resistant compared to susceptible lines of both lupin species with one notable exception: M. persicae numbers were not lower on the resistant variety Tanjil compared to the susceptible variety Tallerack (L. angustifolius). These results suggest that the resistance mechanisms in both lupin species do not affect the selection of hosts by colonizing aphids, but rather are affecting the growth, survival and possibly reproduction of aphids after settling.     

Metabolism of Abscisic Acid and Its Regulation in Xanthium Leaves during and after Water Stress

Metabolism of abscisic acid was compared in stressed and in rehydrated leaf blades of Xanthium strumarium L. Chicago strain that were either detached or left intact on the plant. Under all conditions, phaseic acid was the major metabolite. The high level of phaseic acid that was observed in intact plants 1 day after recovery from stress declined slowly and had not yet reached the prestress level 1 week later. The glucosyl ester of abscisic acid, β-d-glucopyranosyl abscisate, accumulated at a low rate during periods of prolonged stress. Repeated stress-recovery cycles resulted in a gradual increase in the level of the glucosyl ester, which did not decline following relief of stress for at least 34 days. The level of the glucosyl ester of abscisic acid may therefore serve as a cumulative indicator of the water stresses to which a particular leaf has been exposed.     

Abscisic Acid Content and Stomatal Sensitivity to CO(2) in Leaves of Xanthium strumarium L. after Pretreatments in Warm and Cold Growth Chambers

The degree of stomatal sensitivity to CO₂ was positively correlated with the content of abscisic acid of leaves of Xanthium strumarium grown in a greenhouse and then transferred for 24 hours or more to a cold (5/10 C, night/day) or a warm growth chamber (20/23 C). This correlation did not exist in plants kept in the greehouse continuously (high abscisic acid, no CO₂ sensitivity), nor in plants transferred from the cold to the warm chamber (low abscisic acid, high CO₂ sensitivity). The abscisic acid content of leaves was correlated with water content only within narrow limits, if at all. At equal water contents, prechilled leaves contained more abscisic acid than leaves of plants pretreated in the warm chamber. There appear to be at least two compartments for abscisic acid in the leaf.     

Factors Affecting the Abscission of Reproductive Organs in Yellow Lupins (Lupinus luteus L.): III. ENDOGENOUS GROWTH SUBSTANCES IN VIRUS-INFECTED AND HEALTHY PLANTS AND THEIR EFFECT ON ABSCISSION

Extracts of small and mature-size lupin pods yielded four substancesaffecting the growth of wheat-coleoptile sections: one acidpromotor (A), two acid inhibitors(B and X), and one neutralinhibitor(Y). Inhibitor B was extremely active, however, coleoptile sectionsshowed no signs of toxic effects; they resumed growth at a rapidrate after rinsing them and adding ß-indolylaceticand (IAA) to the medium. 1 µg of IAA was required to counteractthe effect of ‘B’ extracted from 230 mg. Of tissue.On an equal fresh weight basis the inhibiting action of ‘B’in lupin pods was 500–1,500 times more potent than thatof ‘inhibitor ß’ in etiolated pea seedlings. Small pods of plants infected with pea-mosaic virus yielded3 times the amount of ‘A’ of healthy plants (equivalentto 1 µg. IAA 0.3 µg. IAA per 25 g. of tissue respectively),and approximately the amount of ‘B’. Mature podsof virus-infected plants again yielded more‘A’,but also 2? times more ‘B’ than pods of healthyplants. Healthy pods yielded more ‘A’ than virus-infectedpods, and there was no difference in ‘X’. A lupin abscission test was developed and the effects of proximaland distal application of -naphthyl acetic acid (NAA) are presented,and discussed with respect to results of other abscission tests. ‘A’ accelerated abscission when applied proximally,and delayed or prevented it when applied distally. ‘B’strongly accelerated abscission when applied in either way.A possible mechanism explaining the abscission-inducing effectof developing pods on later flowers is discussed in terms ofthe substances ‘A’ and ‘B’. The partlyprevented abscission observed on virus-infected plants was foundto agree well with the proposed mechanism.     

Abscisic Aldehyde Is an Intermediate in the Enzymatic Conversion of Xanthoxin to Abscisic Acid in Phaseolus vulgaris L. Leaves

The enzymatic conversion of xanthoxin to abscisic acid by cell-free extracts of Phaseolus vulgaris L. leaves has been found to be a two-step reaction catalyzed by two different enzymes. Xanthoxin was first converted to abscisic aldehyde followed by conversion of the latter to abscisic acid. The enzyme activity catalyzing the synthesis of abscisic aldehyde from xanthoxin (xanthoxin oxidase) was present in cell-free leaf extracts from both wild type and the abscisic acid-deficient molybdopterin cofactor mutant, Az34 (nar2a) of Hordeum vulgare L. However, the enzyme activity catalyzing the synthesis of abscisic acid from abscisic aldehyde (abscisic aldehyde oxidase) was present only in extracts of the wild type and no activity could be detected in either turgid or water stressed leaf extracts of the Az34 mutant. Furthermore, the wilty tomato mutants, sitiens and flacca, which do not accumulate abscisic acid in response to water stress, have been shown to lack abscisic aldehyde oxidase activity. When this enzyme fraction was isolated from leaf extracts of P. vulgaris L. and added to extracts prepared from sitiens and flacca, xanthoxin was converted to abscisic acid. Abscisic aldehyde oxidase has been purified about 145-fold from P. vulgaris L. leaves. It exhibited optimum catalytic activity at pH 7.25 in potassium phosphate buffer.     

Development of microsatellite markers in Lupinus luteus (Fabaceae) and cross-species amplification in other lupine species

? Premise of the study: Microsatellite primers were developed in Lupinus luteus L., an emerging temperate protein crop, to investigate genetic diversity, population structure, and to facilitate the generation of better yellow lupine varieties. ? Methods and Results: Thirteen polymorphic primer sets were evaluated in a European and Eastern European accession collection of L. luteus. The primers amplified di-, tri-, and tetranucleotide repeats with 2-4 alleles per locus. These revealed a moderate to low level of genetic variation, as indicated by an average observed heterozygosity of 0.0126. Select loci also amplified successfully in the closely related species L. hispanicus Boiss. & Reut. and in the New World species L. mutabilis Sweet. ? Conclusions: These results indicate the utility of primers for the study of genetic diversity across L. luteus populations and related lupine species. The use of these microsatellite markers will facilitate the implementation of several molecular breeding strategies in yellow lupine.     

Studies on the distribution of Na and CI in two species of lupin (Lupinus luteus and Lupinus angustifolius) differing in salt tolerance

The distribution of K, Na and CI in various tissues was studied in two species of lupin, Lupinus luteus L. (ex. Portugal) and L. angustifolius L. (cv. Kubesa) under conditions of NaCl-induced salinity stress. L. luteus appeared more tolerant to salt and less effective in excluding Na and Cl from its above-ground parts than L. angustifolius . Electron probe X-ray microanalysis of vacuolar contents of individual root cells of salt-treated L. luteus showed a decreasing gradient of Na and CI contents from the epidermis inwards, but in the inner cortical cells adjacent to the pericycle/endodermis, Na levels were again high while Cl remained low. L. luteus may be inefficient in restricting entry of Na into the shoot because of a limited capacity for Na storage in the vacuoles of root cells. In addition, this species appears to exclude Cl from the vacuoles of inner cortical root cells but not from its symplastic pathway. In the leaves of L. luteus , Cl levels were particularly high towards the lower surface (abaxial side), while Na accumulated in the adaxial side of the leaf. Likewise, different cells of the petiole appeared to place unequal demands on Na and Cl for osmotic adjustment. It is suggested that excess Na in the adaxial side of the leaf and the generally high levels of Na and CI in the foliage may explain the 50% fresh weight stimulation which was observed in L. luteus but not in L. angustifolius at 50 m M NaCl. However, the halophytic response of L. luteus is limited to moderate concentrations of NaCl since 100 m M NaCl caused severe necrosis and leaf abscission.     

Abscisic Acid Refines the Synthesis of Chloroplast Proteins in Maize (Zea mays) in Response to Drought and Light

To better understand abscisic acid (ABA) regulation of the synthesis of chloroplast proteins in maize (Zea mays L.) in response to drought and light, we compared leaf proteome differences between maize ABA-deficient mutant vp5 and corresponding wild-type Vp5 green and etiolated seedlings exposed to drought stress. Proteins extracted from the leaves of Vp5 and vp5 seedlings were used for two-dimensional electrophoresis (2-DE) and subsequent matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). After Coomassie brilliant blue staining, approximately 450 protein spots were reproducibly detected on 2-DE gels. A total of 36 differentially expressed protein spots in response to drought and light were identified using MALDI-TOF MS and their subcellular localization was determined based on the annotation of reviewed accession in UniProt Knowledgebase and the software prediction. As a result, corresponding 13 proteins of the 24 differentially expressed protein spots were definitely localized in chloroplasts and their expression was in an ABA-dependent way, including 6 up-regulated by both drought and light, 5 up-regulated by drought but down-regulated by light, 5 up-regulated by light but down-regulated by drought; 5 proteins down-regulated by drought were mainly those involved in photosynthesis and ATP synthesis. Thus, the results in the present study supported the vital role of ABA in regulating the synthesis of drought- and/or light-induced proteins in maize chloroplasts and would facilitate the functional characterization of ABA-induced chloroplast proteins in C₄ plants.