Molecular and Hormonal Aspects of Drought-Triggered Flower Shedding in Yellow Lupine.

The drought is a crucial environmental factor that determines yielding of many crop species, e.g., Fabaceae, which are a source of valuable proteins for food and feed. Herein, we focused on the events accompanying drought-induced activation of flower abscission zone (AZ)-the structure responsible for flower detachment and, consequently, determining seed production in Lupinus luteus. Therefore, detection of molecular markers regulating this process is an excellent tool in the development of improved drought-resistant cultivars to minimize yield loss. We applied physiological, molecular, biochemical, immunocytochemical, and chromatography methods for a comprehensive examination of changes evoked by drought in the AZ cells. This factor led to significant cellular changes and activated AZ, which consequently increased the flower abortion rate. Simultaneously, drought caused an accumulation of mRNA of genes inflorescence deficient in abscission-like (LlIDL), receptor-like protein kinase HSL (LlHSL), and mitogen-activated protein kinase6 (LlMPK6), encoding succeeding elements of AZ activation pathway. The content of hydrogen peroxide (H2O2), catalase activity, and localization significantly changed which confirmed the appearance of stressful conditions and indicated modifications in the redox balance. Loss of water enhanced transcriptional activity of the abscisic acid (ABA) and ethylene (ET) biosynthesis pathways, which was manifested by elevated expression of zeaxanthin epoxidase (LlZEP), aminocyclopropane-1-carboxylic acid synthase (LlACS), and aminocyclopropane-1-carboxylic acid oxidase (LlACO) genes. Accordingly, both ABA and ET precursors were highly abundant in AZ cells. Our study provides information about several new potential markers of early response on water loss, which can help to elucidate the mechanisms that control plant response to drought, and gives a useful basis for breeders and agronomists to enhance tolerance of crops against the stress.

Metabolic diversification of nitrogen-containing metabolites by the expression of a heterologous lysine decarboxylase gene in Arabidopsis.

Lysine decarboxylase converts l-lysine to cadaverine as a branching point for the biosynthesis of plant Lys-derived alkaloids. Although cadaverine contributes towards the biosynthesis of Lys-derived alkaloids, its catabolism, including metabolic intermediates and the enzymes involved, is not known. Here, we generated transgenic Arabidopsis lines by expressing an exogenous lysine/ornithine decarboxylase gene from Lupinus angustifolius (La-L/ODC) and identified cadaverine-derived metabolites as the products of the emerged biosynthetic pathway. Through untargeted metabolic profiling, we observed the upregulation of polyamine metabolism, phenylpropanoid biosynthesis and the biosynthesis of several Lys-derived alkaloids in the transgenic lines. Moreover, we found several cadaverine-derived metabolites specifically detected in the transgenic lines compared with the non-transformed control. Among these, three specific metabolites were identified and confirmed as 5-aminopentanal, 5-aminopentanoate and δ-valerolactam. Cadaverine catabolism in a representative transgenic line (DC29) was traced by feeding stable isotope-labeled [α-15 N]- or [ε-15 N]-l-lysine. Our results show similar 15 N incorporation ratios from both isotopomers for the specific metabolite features identified, indicating that these metabolites were synthesized via the symmetric structure of cadaverine. We propose biosynthetic pathways for the metabolites on the basis of metabolite chemistry and enzymes known or identified through catalyzing specific biochemical reactions in this study. Our study shows that this pool of enzymes with promiscuous activities is the driving force for metabolite diversification in plants. Thus, this study not only provides valuable information for understanding the catabolic mechanism of cadaverine but also demonstrates that cadaverine accumulation is one of the factors to expand plant chemodiversity, which may lead to the emergence of Lys-derived alkaloid biosynthesis.

Long-Term Effects of Cold on Growth, Development and Yield of Narrow-Leaf Lupine May Be Alleviated by Seed Hydropriming or Butenolide.

In this article, the effects of cold on the development of Lupine angustifolius and the possibility of mitigating it, via seed hydropriming or pre-treatment with butenolide (10-6 M⁻10-4 M), are investigated in two cultivars, differing in their ability to germinate at low temperature. Physiological background of plant development after cold stress was investigated in imbibed seeds. For the first four weeks, the seedlings grew at 7 °C or 13 °C. Seeds well germinating at 7 °C demonstrated higher activity of α-amylase and higher levels of gibberellins, IAA and kinetin. Germination ability at low temperature correlated with dehydrogenase activity and membrane permeability. Seed pre-treatment improved germination at low temperature by decreasing abscisic acid content. Seed hydropriming alleviated cold effects on plant development rate and yield, while butenolide accelerated vegetative development but delayed the generative phase. Potential seed yield may be predicted based on the seed germination vigour and the photosynthetic efficiency measured before flowering.

Legume isoflavone synthase genes have evolved by whole-genome and local duplications yielding transcriptionally active paralogs.

Isoflavone synthase (IFS) is the key enzyme of isoflavonoid biosynthesis. IFS genes were identified in numerous species, although their evolutionary patterns have not yet been reconstructed. To address this issue, we performed structural and functional genomic analysis. Narrow leafed lupin, Lupinus angustifolius L., was used as a reference species for the genus, because it has the most developed molecular tools available. Nuclear genome BAC library clones carrying IFS homologs were localized by linkage mapping and fluorescence in situ hybridization in three chromosome pairs. Annotation of BAC, scaffold and transcriptome sequences confirmed the presence of three full-length IFS genes in the genome. Microsynteny analysis and Bayesian inference provided clear evidence that IFS genes in legumes have evolved by lineage-specific whole-genome and tandem duplications. Gene expression profiling and RNA-seq data mining showed that the vast majority of legume IFS copies have maintained their transcriptional activity. L. angustifolius IFS homologs exhibited organ-specific expression patterns similar to those observed in other Papilionoideae. Duplicated lupin IFS homologs retained non-negligible levels of substitutions in conserved motifs, putatively due to positive selection acting during early evolution of the genus, before the whole-genome duplication. Strong purifying selection preserved newly arisen IFS duplicates from further nonsynonymous changes.

Levofloxacin is phytotoxic and modifies the protein profile of lupin seedlings.

The toxicity of levofloxacin to yellow lupin plants was evaluated in this study. Recommended indexes of plant (roots and shoots) growth were determined and new indexes were proposed which better characterise the phytotoxicity of levofloxacin. These were, in particular, the activity of antioxidative enzymes, the content of free radicals, as well as the root protein content and the root protein profile. The results showed that levofloxacin considerably affected EC50, measured as the activity of catalase in roots, and leaves (1.05 and 0.069 mM, respectively). The activity of peroxidase in the roots and the dry weight of seedlings were the least sensitive parameters (EC50 was 1.8 and 1.76 mM, respectively). Units of toxicity clearly showed that the activity of catalase is a better measure of toxicity for low concentrations of the drug, and it is a better index of plant physiological state than the morphological parameters of seedlings. Moreover, levofloxacin changed the location of free radicals and the protein profile in plants. The changes in location of reactive oxygen species in roots were an important symptom of the drug toxicity to lupin seedlings. Our results have shown that the toxicity of levofloxacin was manifested mainly by changes in the protein profile. The content of the glyceraldehyde-3-phosphate dehydrogenase, 14-3-3-like protein A, expansin-B3-like precursor, fructose-bisphosphate aldolase, lipoxygenase, nucleotide-binding subunit of vacuolar ATPase and pyruvate dehydrogenase were found to decrease. On the other hand, plant exposure to levofloxacin resulted in an increase in the content of enolase, protein LlR18A, class III chitinase, ascorbate peroxidase, aspartate aminotransferase, alcohol dehydrogenase 1, leghemoglobin reductase-like 17 and heat shock cognate protein 80-like.

Structural basis of the lack of endo-glucanase inhibitory activity of Lupinus albus γ-conglutin.

Lupin γ-conglutin and soybean BG7S are two legume seed proteins strongly similar to plant endo-ß-glucanases inhibitors acting against fungal GH11 and GH12 glycoside hydrolase. However these proteins lack inhibitory activity. Here we describe the conversion of lupin γ-conglutin to an active inhibitor of endo-ß-glucanases belonging to GH11 family. A set of γ-conglutin mutants was designed and expressed in Pichia pastoris, along with the wild-type protein. Unexpectedly, this latter was able to inhibit a GH11 enzyme, but not GH12, whereas the mutants were able to modulate the inhibition capacity. In lupin, γ-conglutin is naturally cleaved in two subunits, whereas in P. pastoris it is not. The lack of proteolytic cleavage is one of the reasons at the basis of the inhibitory activity of recombinant γ-conglutin. The results provide new insights about structural features at the basis of the lack of inhibitory activity of wild-type γ-conglutin and its legume homologues.

Adenylylsulfate-ammonia adenylyltransferase activity is another inherent property of Fhit proteins.

Fhits (fragile histidine triad proteins) occur in eukaryotes but their function is largely unknown, although human Fhit is believed to act as a tumour suppressor. Fhits also exhibit dinucleoside triphosphatase, adenylylsulfatase and nucleoside phosphoramidase activities that in each case yield nucleoside 5'-monophosphate as a product. Due to the dinucleoside triphosphatase activity, Fhits may also be involved in mRNA decapping. In the present study, we demonstrate Fhit-catalysed ammonolysis of adenosine 5'-phosphosulfate, which results in the formation of adenosine 5'-phosphoramidate. This reaction has previously been associated with adenylylsulfate-ammonia adenylyltransferase (EC 2.7.7.51). Our finding shows that the capacity to catalyse ammonolysis is another inherent property of Fhits. Basic kinetic parameters and substrate specificity of this reaction catalysed by human Fhit are presented.

Immunolocalization of dually phosphorylated MAPKs in dividing root meristem cells of Vicia faba, Pisum sativum, Lupinus luteus and Lycopersicon esculentum.

KEY MESSAGE: In plants, phosphorylated MAPKs display constitutive nuclear localization; however, not all studied plant species show co-localization of activated MAPKs to mitotic microtubules. The mitogen-activated protein kinase (MAPK) signaling pathway is involved not only in the cellular response to biotic and abiotic stress but also in the regulation of cell cycle and plant development. The role of MAPKs in the formation of a mitotic spindle has been widely studied and the MAPK signaling pathway was found to be indispensable for the unperturbed course of cell division. Here we show cellular localization of activated MAPKs (dually phosphorylated at their TXY motifs) in both interphase and mitotic root meristem cells of Lupinus luteus, Pisum sativum, Vicia faba (Fabaceae) and Lycopersicon esculentum (Solanaceae). Nuclear localization of activated MAPKs has been found in all species. Co-localization of these kinases to mitotic microtubules was most evident in L. esculentum, while only about 50% of mitotic cells in the root meristems of P. sativum and V. faba displayed activated MAPKs localized to microtubules during mitosis. Unexpectedly, no evident immunofluorescence signals at spindle microtubules and phragmoplast were noted in L. luteus. Considering immunocytochemical analyses and studies on the impact of FR180204 (an inhibitor of animal ERK1/2) on mitotic cells, we hypothesize that MAPKs may not play prominent role in the regulation of microtubule dynamics in all plant species.

Lipoxygenase activity in different species of sweet lupin (Lupinus L.) seeds and flakes.

Lipoxygenase (LOX)-catalysed degradation of polyunsaturated fatty acids is supposed to be a major cause of undesirable off-flavour development in legumes. In the present study, a photometric LOX assay including adequate sample workup was adapted to lupin seeds, kernels and flakes, respectively. Optimum reaction conditions were at pH 7.5 using a phosphate buffer concentration of 150 mmol l(-1) without the addition of sodium chloride. The LOX activities of different lupin species and varieties were compared. Significant variations among the species and varieties ranging from 50 to 1004 units mg(-1) protein were determined, being significantly lower than soybean LOX activity. Hulling and flaking of the seeds resulted in a 15% increase of LOX activity. In contrast to soy and other legumes, LOX from lupin only converted free fatty acids, whereas trilinolein and ß-carotene were not oxidised. Consequently, according to the established classification, lupin LOX activity may be assigned to the LOX type-1, which, to the best of our knowledge, was demonstrated for the first time.

Expression of novel cytosolic malate dehydrogenases (cMDH) in Lupinus angustifolius nodules during phosphorus starvation.

During P deficiency, the increased activity of malate dehydrogenase (MDH, EC 1.1.1.37) can lead to malate accumulation. Cytosolic- and nodule-enhanced MDH (cMDH and neMDH, respectively) are known isoforms, which contribute to MDH activity in root nodules. The aim of this study was to investigate the role of the cMDH isoforms in nodule malate supply under P deficiency. Nodulated lupins (Lupinus angustifolius var. Tanjil) were hydroponically grown at adequate P (+P) or low P (-P). Total P concentration in nodules decreased under P deficiency, which coincided with an increase in total MDH activity. A consequence of higher MDH activity was the enhanced accumulation of malate derived from dark CO2 fixation via PEPC and not from pyruvate. Although no measurable neMDH presence could be detected via PCR, gene-specific primers detected two 1kb amplicons of cMDH, designated LangMDH1 (corresponding to +P, HQ690186) and LangMDH2 (corresponding to -P, HQ690187), respectively. Sequencing analyses of these cMDH amplicons showed them to be 96% identical on an amino acid level. There was a high degree of diversification between proteins detected in this study and other known MDH proteins, particularly those from other leguminous plants. Enhanced malate synthesis in P-deficient nodules was achieved via increased anaplerotic CO2 fixation and subsequent higher MDH activities. Novel isoforms of cytosolic MDH may be involved, as shown by gene expression of specific genes under P deficiency.

Short-term supply of elevated phosphate alters the belowground carbon allocation costs and functions of lupin cluster roots and nodules.

The legume Lupinus albus is able to survive under low nutrient conditions due to the presence of two specialized below ground organs for the acquisition of nitrogen and phosphate, respectively.In this regard, cluster roots increase phosphate uptake and root nodules acquire atmospheric N2via biological nitrogen fixation(BNF). Although these organs normally tolerate low phosphate conditions, very little is known about their physiological and metabolic flexibility during short-term changes in phosphate supply. The aim of this investigation was therefore to determine the physiological and metabolic flexibility of these organs during short-term supply of elevated phosphate nutrition. L. albus was cultivated in sand culture for 4 weeks at 0.1 mM phosphate supply, and then supplied with 2 mM phosphate for 2 weeks. Short-term elevated phosphate supply caused increased allocation of carbon and respiratory costs to nodules, at the expense of cluster root function. This alteration was also reflected in the increase in nodule enzyme activities related to organic acid synthesis, such as Phosphoenol-pyruvate Carboxylase (PEPC), Pyruvate Kinase (PK), Malate Dehydrogenase(NADH-MDH) and Malic Enzyme (ME). In cluster roots, elevated phosphate conditions caused a decline in these organic acid synthesizing enzymes. Phosphate recycling via Acid Phosphatase (APase),declined in nodules with elevated phosphate supply, but increased in cluster roots. Our findings suggest that during short-term elevated phosphate supply, there is a great degree of physiological and metabolic flexibility in lupin nutrient acquiring structures, and that these changes are related to the altered physiology of these organs [corrected].

The alternative respiratory pathway mediates carboxylate synthesis in white lupin cluster roots under phosphorus deprivation.

Plant adaptations associated with a high efficiency of phosphorus (P) acquisition can be used to increase productivity and sustainability in a world with a growing population and decreasing rock phosphate reserves. White lupin (Lupinus albus) produces cluster roots that release carboxylates to efficiently mobilize P from P-sorbing soils. It has been hypothesized that an increase in the activity of the alternative oxidase (AOX) would allow for the mitochondrial oxidation of NAD(P)H produced during citrate synthesis in cluster roots at a developmental stage when there is a low demand for ATP. We used the oxygen-isotope fractionation technique to study the in vivo respiratory activities of the cytochrome oxidase pathway (COP) and the alternative oxidase pathway (AOP) in different root sections of white lupins grown hydroponically with and without P. In parallel, AOX protein levels and internal carboxylate concentrations were determined in cluster and non-cluster roots. Higher in vivo AOP activity was measured in cluster roots when malate and citrate concentrations were also high, thus confirming our hypothesis. AOX protein levels were not always correlated with in vivo AOP activity, suggesting post-translational regulation of AOX.

Copper microlocalisation and changes in leaf morphology, chloroplast ultrastructure and antioxidative response in white lupin and soybean grown in copper excess.

The microlocalisation of Cu was examined in the leaves of white lupin and soybean grown hydroponically in the presence of 1.6 (control) or 192 µM (excess) Cu, along with its effect on leaf morphology, (ultra)structure and the antioxidative response. The 192 µM dose led to a reduction in the total leaf area and leaf thickness in both species, although more strongly so in white lupin. In the latter species it was also associated with smaller spongy parenchyma cells, and smaller spaces between them, while in the soybean it more strongly reduced the size of the palisade parenchyma and epidermal cells. Energy-dispersive X-ray microanalysis showed that under Cu excess the metal was mainly localised inside the spongy parenchyma cells of the white lupin leaves, and in the lower epidermis cell walls in those of the soybean. Cu excess also promoted ultrastructural chloroplast alterations, reducing the photosynthetic capacity index and the green area of the leaves, especially in the soybean. Despite this, soybean appeared to be more tolerant to Cu excess than white lupin, because soybean displayed (1) lower accumulation of Cu in the leaves, (2) enhanced microlocalisation of Cu in the cell walls and (3) greater levels of induced total -SH content and superoxide dismutase and catalase activities that are expected for better antioxidative responses.

ß-N-Acetylhexosaminidase involvement in α-conglutin mobilization in Lupinus albus.

Glycosylation is an important post-translational modification involved in the modulation of a wide variety of cellular processes. Because glycosydases are central, the aim of this study was to investigate the glycosyl activity present in the cotyledons of the seeds of an important crop legume, Lupinus albus, as well as potential natural substrates of the detected enzymes. The glycosyl activity detected in the cotyledons beginning at seed imbibition and continuing until 9 days after, was due to a ß-N-acetylhexosaminidase (ß-NAHase), which was molecularly and biochemically characterized after purification. Two isoenzymes with molecular masses of 64 and 61 kDa were detected, each having five isoenzymes with pIs 5.3-5.6. The 64 and 61 kDa isoenzymes had the same protein core showing different degrees of glycosylation. The N-terminal sequence of the enzyme protein core was determined [VDSEDLI(EN)AFKIYVEDDNEHLQGSVD] and to our knowledge, is the first reported protein sequence from a plant ß-NAHase. L. albus ß-NAHase had Km values of 2.59 mM and 2.94 mM and V values of 18.40 µM min(-1) and 2.73 µM min(-1), for pNP-GlcNAc and pNP-GalNAc, an optimum pH of 5.0 and 4.0 and temperature of 50 °C and 60 °C were detected toward pNP-GlcNAc and pNP-GalNAc. In the presence of AgNO3, CoCl2, CuSO4, FeCl3, CdCl2 and ZnCl2 the enzymatic activity decreased more than 50%, and when in the presence of sugars, an activity reduction of no more than 25% was observed. A physiological role for ß-NAHase in L. albus storage protein mobilization was investigated. ß-NAHase has already been implicated in several biological processes, namely in glycoprotein processing during seed germination and seedling growth. However, the natural substrates used by this enzyme are not yet completely clarified. By gathering in vivo and in vitro data for ß-NAHase activity together with globulin degradation, we suggest that L. albus ß-NAHase is involved in the mobilization of storage protein degradation, with α-conglutin being a potential natural substrate for this enzyme.

The proteome of exudates from germinating Lupinus albus seeds is secreted through a selective dual-step process and contains proteins involved in plant defence.

The general knowledge of defence activity during the first steps of seed germination is still largely incomplete. The present study focused on the proteins released in the exudates of germinating white lupin seeds. During the first 24 h, a release of proteins was observed. Initially (i.e. during the first 12 h), the proteins found in exudates reflected the composition of the seed, indicating a passive extrusion of pre-formed proteins. Subsequently, when the rate of protein release was at its highest, the composition of the released proteome changed drastically. This transition occurred in a short time, indicating that more selective and regulated events, such as secretory processes, took place soon after the onset of germination. The present study considered: (a) the characterization of the proteome accumulated in the germinating medium collected after the appearance of the post-extrusion events; (b) the biosynthetic origin and the modalities that are the basis of protein release outside the seeds; and (c) an assessment of antifungal activity of these exudates. The most represented protein in the exudate was chitinase, which was synthesized de novo. The other proteins are involved in the cellular mechanisms responding to stress events, including biotic ones. This exudate was effectively able to inhibit fungal growth. The results of the present study indicate that seed exudation is a dual-step process that leads to the secretion of selected proteins and thus is not a result of passive leakage. The released proteome is involved in protecting the spermosphere environment and thus may act as first defence against pathogens.

Lysine decarboxylase catalyzes the first step of quinolizidine alkaloid biosynthesis and coevolved with alkaloid production in leguminosae.

Lysine decarboxylase (LDC) catalyzes the first-step in the biosynthetic pathway of quinolizidine alkaloids (QAs), which form a distinct, large family of plant alkaloids. A cDNA of lysine/ornithine decarboxylase (L/ODC) was isolated by differential transcript screening in QA-producing and nonproducing cultivars of Lupinus angustifolius. We also obtained L/ODC cDNAs from four other QA-producing plants, Sophora flavescens, Echinosophora koreensis, Thermopsis chinensis, and Baptisia australis. These L/ODCs form a phylogenetically distinct subclade in the family of plant ornithine decarboxylases. Recombinant L/ODCs from QA-producing plants preferentially or equally catalyzed the decarboxylation of L-lysine and L-ornithine. L. angustifolius L/ODC (La-L/ODC) was found to be localized in chloroplasts, as suggested by the transient expression of a fusion protein of La-L/ODC fused to the N terminus of green fluorescent protein in Arabidopsis thaliana. Transgenic tobacco (Nicotiana tabacum) suspension cells and hairy roots produced enhanced levels of cadaverine-derived alkaloids, and transgenic Arabidopsis plants expressing (La-L/ODC) produced enhanced levels of cadaverine, indicating the involvement of this enzyme in lysine decarboxylation to form cadaverine. Site-directed mutagenesis and protein modeling studies revealed a structural basis for preferential LDC activity, suggesting an evolutionary implication of L/ODC in the QA-producing plants.

Characterization of an isoflavonoid-specific prenyltransferase from Lupinus albus.

Prenylated flavonoids and isoflavonoids possess antimicrobial activity against fungal pathogens of plants. However, only a few plant flavonoid and isoflavonoid prenyltransferase genes have been identified to date. In this study, an isoflavonoid prenyltransferase gene, designated as LaPT1, was identified from white lupin (Lupinus albus). The deduced protein sequence of LaPT1 shared high homologies with known flavonoid and isoflavonoid prenyltransferases. The LaPT1 gene was mainly expressed in roots, a major site for constitutive accumulation of prenylated isoflavones in white lupin. LaPT1 is predicted to be a membrane-bound protein with nine transmembrane regions and conserved functional domains similar to other flavonoid and isoflavonoid prenyltransferases; it has a predicted chloroplast transit peptide and is plastid localized. A microsomal fraction containing recombinant LaPT1 prenylated the isoflavone genistein at the B-ring 3' position to produce isowighteone. The enzyme is also active with 2'-hydroxygenistein but has no activity with other flavonoid substrates. The apparent K(m) of recombinant LaPT1 for the dimethylallyl diphosphate prenyl donor is in a similar range to that of other flavonoid prenyltransferases, but the apparent catalytic efficiency with genistein is considerably higher. Removal of the transit peptide increased the apparent overall activity but also increased the K(m). Medicago truncatula hairy roots expressing LaPT1 accumulated isowighteone, a compound that is not naturally produced in this species, indicating a strategy for metabolic engineering of novel antimicrobial compounds in legumes.

Identification in lupin seed of a serine-endopeptidase activity cleaving between twin arginine pairs and causing limited proteolysis of seed storage proteins.

The occurrence of twin-arginine motifs (-R-R-) in the amino acid sequences of animal pro-proteins frequently defines the cleavage site(s) for their structural/functional maturation. No information is available on the presence and possible biological meaning of these motifs in the seed storage proteins. In this work, a novel endopeptidase activity with cleavage specificity to twin-arginine pairs has been detected in mature dry Lupinus albus seeds. The endopeptidase was tested with a number of endogenous and exogenous protein substrates, which were selected according to the presence of one or more twin-arginine residue motifs in their amino acid sequences. The observed hydrolysis patterns were limited and highly specific. Partial proteolysis led to stable polypeptide fragments that were characterized by 1- and 2-D electrophoresis. Selected polypeptides were submitted to N-terminal amino acid sequencing and mass spectrometry analyses. These approaches, supported by bioinformatic analysis of the available sequences, allowed the conclusion that the polypeptide cleavage events had occurred at the peptide bonds comprised between twin-arginine residue pairs with all tested protein substrates. The endopeptidase activity was inhibited by 4-(2-AminoEthyl)Benzene-Sulphonyl Fluoride hydrochloride (AEBSF), leupeptin, and serine proteinase protein inhibitors, while it was not affected by pepstatin, trans-Epoxysuccinyl-L-leucylamido(4-guanidino)butane (E64), and ethylenediaminetetraacetic acid (EDTA), thus qualifying the Arg-Arg cleaving enzyme as a serine endopeptidase. The structural features of storage proteins from lupin and other legume seeds strongly support the hypothesis that the occurrence of an endopeptidase activity cleaving -R-R- bonds may be functional to facilitate their degradation at germination and possibly generate polypeptide fragments with specific biological activity.

Role of asparaginase variable loop at the carboxyl terminal of the alpha subunit in the determination of substrate preference in plants.

Structural determinants responsible for the substrate preference of the potassium-independent (ASPGA1) and -dependent (ASPGB1) asparaginases from Arabidopsis thaliana have been investigated. Like ASPGA1, ASPGB1 was found to be catalytically active with both L: -Asn and ß-Asp-His as substrates, contrary to a previous report. However, ASPGB1 had a 45-fold higher specific activity with Asn as substrate than ASPGA1. A divergent sequence between the two enzymes forms a variable loop at the C-terminal of the alpha subunit. The results of dynamic simulations have previously implicated a movement of the C-terminus in the allosteric transduction of K(+)-binding at the surface of LjNSE1 asparaginase. In the crystal structure of Lupinus luteus asparaginase, most residues in this segment cannot be visualized due to a weak electron density. Exchanging the variable loop in ASPGA1 with that from ASPGB1 increased the affinity for Asn, with a 320-fold reduction in K (m) value. Homology modeling identified a residue specific to ASPGB1, Phe(162), preceding the variable loop, whose side chain is located in proximity to the beta-carboxylate group of the product aspartate, and to Gly(246), a residue participating in an oxyanion hole which stabilizes a negative charge forming on the side chain oxygen of asparagine during catalysis. Replacement with the corresponding leucine from ASPGA1 specifically lowered the V (max) value with Asn as substrate by 8.4-fold.

Sucrose controls storage lipid breakdown on gene expression level in germinating yellow lupine (Lupinus luteus L.) seeds.

This study revealed that cytosolic aconitase (ACO, EC 4.2.1.3) and isocitrate lyase (ICL, EC 4.1.3.1, marker of the glyoxylate cycle) are active in germinating protein seeds of yellow lupine. The glyoxylate cycle seems to function not only in the storage tissues of food-storage organs, but also in embryonic tissue of growing embryo axes. Sucrose (60mM) added to the medium of in vitro culture of embryo axes and cotyledons decreased activity of lipase (LIP, EC 3.1.1.3) and activity of glutamate dehydrogenase (NADH-GDH, EC 1.4.1.2). The opposite effect was caused by sucrose on activity of cytosolic ACO, ICL as well as NADP(+)-dependent (EC 1.1.1.42) and NAD(+)-dependent (EC 1.1.1.41) isocitrate dehydrogenase (NADP-IDH and NAD-IDH, respectively); activity of these enzymes was clearly stimulated by sucrose. Changes in the activity of LIP, ACO, NADP-IDH, and NAD-IDH caused by sucrose were based on modifications in gene expression because corresponding changes in the enzyme activities and in the mRNA levels were observed. The significance of cytosolic ACO and NADP-IDH in carbon flow from storage lipid to amino acids, as well as the peculiar features of storage lipid breakdown during germination of lupine seeds are discussed.