Search

Background: Peas are an inexpensive yet nutritious and sustainable source of protein. However, it is challenging to incorporate pea proteins into food formulations owing to their beany or green off-flavours and their limited water solubility., Results: Vacuum microwave dehydration (VMD) of pea protein with an initial moisture content of 425% (dry basis, db) at 2 W g -1 specific microwave energy and 200 Torr vacuum level for 88 min led to an 83% reduction in total volatile compound concentration. VMD processing at high initial moisture contents facilitated the Maillard reaction, enhancing the extent of protein cross-linking, leading to a marked decrease in soluble protein content, to 11 g kg -1 . Reducing the initial moisture content to 56% db greatly retained protein solubility (112-113 g kg -1 ), but it only led to a minor reduction in total volatile compound concentration (2-11% reduction). A high microwave energy (20 W g -1 )-short time (2 min) treatment at 200 Torr vacuum level was found optimal, reducing both volatile levels and soluble protein content by ~50%., Conclusion: Evidently, it is difficult to employ VMD without reduction of pea protein solubility and corresponding changing in functionality. Yet, if optimized, VMD has the capability to decrease volatile concentrations while retaining protein solubility. Future sensory analysis should be conducted to determine whether the aforementioned reductions in total volatile compound concentration may have a notable effect on consumer palatability. © 2020 Society of Chemical Industry., (© 2020 Society of Chemical Industry.)

Plant responses to light involve a complex network of interactions among multiple plant hormones. In a screen for mutants showing altered photomorphogenesis under red light, we identified a mutant with dramatically enhanced leaf expansion and delayed petal senescence. We show that this mutant exhibits reduced sensitivity to ethylene and carries a nonsense mutation in the single pea (Pisum sativum) ortholog of the ethylene signaling gene ETHYLENE INSENSITIVE2 (EIN2). Consistent with this observation, the ein2 mutation rescues the previously described effects of ethylene overproduction in mature phytochrome-deficient plants. In seedlings, ein2 confers a marked increase in leaf expansion under monochromatic red, far-red, or blue light, and interaction with phytochromeA, phytochromeB, and long1 mutants confirms that ein2 enhances both phytochrome- and cryptochrome-dependent responses in a LONG1-dependent manner. In contrast, minimal effects of ein2 on seedling development in darkness or high-irradiance white light show that ethylene is not limiting for development under these conditions. These results indicate that ethylene signaling constrains leaf expansion during deetiolation in pea and provide further evidence that down-regulation of ethylene production may be an important component mechanism in the broader control of photomorphogenic development by phytochrome and cryptochrome., (© 2015 American Society of Plant Biologists. All Rights Reserved.)

The pea (Pisum sativum) tetrameric short-chain alcohol dehydrogenase-like protein (SAD) family consists of at least three highly similar members (SAD-A, -B, and -C). According to mRNA data, environmental stimuli induce SAD expression. The aim of this study was to characterize the SAD proteins by examining their catalytic function, distribution in pea, and induction in different tissues. In enzyme activity assays using a range of potential substrates, the SAD-C enzyme was shown to reduce one- or two-ring-membered quinones lacking long hydrophobic hydrocarbon tails. Immunological assays using a specific antiserum against the protein demonstrated that different tissues and cell types contain small amounts of SAD protein that was predominantly located within epidermal or subepidermal cells and around vascular tissue. Particularly high local concentrations were observed in the protoderm of the seed cotyledonary axis. Two bow-shaped rows of cells in the ovary and the placental surface facing the ovule also exhibited considerable SAD staining. Ultraviolet-B irradiation led to increased staining in epidermal and subepidermal cells of leaves and stems. The different localization patterns of SAD suggest functions both in development and in responses to environmental stimuli. Finally, the pea SAD-C promoter was shown to confer heterologous wound-induced expression in Arabidopsis (Arabidopsis thaliana), which confirmed that the inducibility of its expression is regulated at the transcriptional level.

The contribution to reduction of oxygen by ferredoxin (Fd) to the overall reduction of oxygen in isolated pea thylakoids was studied in the presence of Fd versus Fd + NADP(+). The overall rate of electron transport was measured using a determination of Photosystem II quantum yield from chlorophyll fluorescence parameters, and the rate of oxidation of Fd was measured from the light-induced redox changes of Fd. At low light intensity, increasing Fd concentration from 5 to 30 microM in the absence of NADP(+) increased the proportion of oxygen reduction by Fd from 25-35 to 40-60% in different experiments. This proportion decreased with increasing light intensity. When NADP(+) was added in the presence of 15 microM Fd, which was optimal for the NADP(+) reduction rate, the participation of Fd in the reduction of oxygen was low, no more than 10%, and it also decreased with increasing light intensity. At high light intensity, the overall oxygen reduction rates in the presence of Fd + NADP(+) and in the presence of Fd alone were comparable. The significance of reduction of dioxygen either by water-soluble Fd or by the membrane-bound carriers of the photosynthetic electron transport chain for redox signaling under different light intensities is discussed.

Photosystem I (PSI) is severely damaged by chilling at 4 degrees C in low light, especially in the chilling sensitive plant cucumber. To investigate the early events in PSI photoinhibition, we examined structural changes in the level of pigment-protein complexes in cucumber leaves in comparison with pea leaves. The complexes were separated on a native green gel and an increase in the intensity of a band was observed only in light-chilled cucumber leaves. The 77 K fluorescence emission spectrum of this green band indicated that the band was mainly composed of PSI with light-harvesting complex I. Each lane was cut from the green gel and separated on a fully denaturing SDS-PAGE in the second dimension. The new green gel band observed after light-chilling in cucumber leaves lacked 19, 18, and 16.5 kDa polypeptides. These results suggest that light-chilling facilitates the release of three peripheral polypeptides as an early event of chilling stress in vivo, which results in the inactivation of PSI in intact cucumber leaves.

Ran/TC4 is the only known member of the family of small GTP-binding proteins primarily localized inside the nucleus. We cloned a pea Ran gene (PsRan1) and characterized its expression in tissues, and under different light sources. PsRan1 is a member of a highly homologous multigene family, and it encodes a protein containing plant-specific amino acids in its sequence. It is ubiquitously expressed in pea tissues with high expression in radicles. The amount of total mRNA transcripts representing multiple Ran family members increased in response to very low-fluence R, while the amount of mRNA transcript encoding PsRan1 specifically was not affected by various light treatments. In addition, Ran genes in Arabidopsis were also differentially expressed in various mutants defective in phytochromes or the light-responding HY5 protein, such as phyA, phyB, and hy5. AtRan1 and AtRan3 gene expression was significantly reduced in the phyA mutant background compared to that in Ler-0 wild type plants. AtRan1 expression was also decreased in the phyB background. In contrast, the expression of AtRan2 did not vary in the hy5 and phytochrome mutant backgrounds examined. Interestingly, expression of AtRan1 was significantly reduced in hy5 plants, while AtRan3 expression was increased in the same plants. From these results, we conclude that Ran gene expression is differentially regulated by various light sources and phytochrome-mediated signaling pathways.

NADPH is an essential electron donor in numerous biosynthetic and detoxification reactions. In animal, yeast and bacteria, the NADP-dependent isocitrate dehydrogenase (NADP-ICDH), which catalyzes the production of NADPH, is being recognized as an essential component of the antioxidative defence mechanisms. In plant cells, there is little information on the antioxidant properties of NADP-ICDH. Using a pea cDNA lambdagt11 library, the full-length cDNA of a NADP-ICDH was obtained. In pea leaves, the analyses of activity, protein and transcript expression of NADP-ICDH under six different abiotic stress conditions (CL, continuous light, HLI, high light intensity, D, continuous dark, LT, low-temperature HT, high-temperature and W, mechanical wounding) revealed a differential regulation at transcriptional and post-translational level depending on the abiotic stress. The activity and protein expression of NADP-ICDH and catalase increased only under HLI but the NADP-ICDH transcripts were up-regulated by cold stress (70%) and W (40%). Under the same conditions, the transcript analysis of glutathione reductase (GR), monodehydroascorbate reductase (MDAR) and ascorbate peroxidase (APX), key components of the antioxidative ascorbate-glutathione cycle, showed similar inductions. These data indicate that in pea plants the cytosolic NADP-ICDH shows a differential response, at mRNA and activity level, depending on the type of abiotic stress and suggests that this dehydrogenase could have a protective antioxidant role against certain environmental stresses in plants.

Pea seeds (Pisum sativum L.) from the seed lot with 80% germinability were separated in fractions according to room temperature phosphorescence: strong seeds were assigned to fraction I, and weak seeds formed to fraction II. During imbibition, the seeds of fraction II exhibited twofold higher rates of water uptake and experienced hypoxia. Some of these seeds suffocated from hypoxia, and other seeds produced seedlings with morphological defects (such seeds were considered incapable of germination). One week after irradiation with the dose of 3 Gy, germination percentage decreased to 45%, which was caused by the increase of number of weak seeds. The germinability of seeds subjected to gamma-irradiation at doses of 7 and 10 Gy was similar to that of control seeds. In these sub-lots, there appeared so-called "improved" seeds, which were similar to non-irradiated seeds in terms of phosphorescence level, the rate of water uptake and germination percentage. It was shown with the use of PCMB that the difference in the rates of water uptake by seeds of fraction I and II depended on the permeability of cell membranes. The permeability was determined by the state of aquaporins ("open"-"closed"). The experiments with phosphatase inhibitor (NaF) shown that in seeds irradiated with dose of 3 Gy (fraction II), the mechanism of aquaporins closing was broken (phosphatase was inactivated). In "improved" seeds (after irradiation with dose of 10 Gy), aquaporins were closed irreversibly in air-dry state, when aquaporin dephosphorylation was unlikely. It was concluded that the abnormal increase (following the initially decrease) in germination of pea seeds after irradiation can be explained without invoking the hypothesis on hyper function of reparatory mechanism of at low doses of irradiation.

The plant light-harvesting complex of photosystem II (LHC-II) collects and transmits solar energy for photosynthesis in chloroplast membranes and has essential roles in regulation of photosynthesis and in photoprotection. The 2.5 A structure of pea LHC-II determined by X-ray crystallography of stacked two-dimensional crystals shows how membranes interact to form chloroplast grana, and reveals the mutual arrangement of 42 chlorophylls a and b, 12 carotenoids and six lipids in the LHC-II trimer. Spectral assignment of individual chlorophylls indicates the flow of energy in the complex and the mechanism of photoprotection in two close chlorophyll a-lutein pairs. We propose a simple mechanism for the xanthophyll-related, slow component of nonphotochemical quenching in LHC-II, by which excess energy is transferred to a zeaxanthin replacing violaxanthin in its binding site, and dissipated as heat. Our structure shows the complex in a quenched state, which may be relevant for the rapid, pH-induced component of nonphotochemical quenching.

DNA fragments containing the 5' promoter regions of the Pisum sativum sadA and sadC genes were amplified from genomic DNA, cloned and sequenced. These sequences contain a number of conserved cis-acting elements, which are potentially involved in stress-induced transcription of the sad genes. To determine whether any of the identified elements are active in binding nuclear factors in vitro, 11 60-bp overlapping (by 30 bp) DNA probe fragments covering the proximal sadC promoter sequence (360 bp) were used in electrophoretic mobility shift assays with competition. Binding activities were compared in nuclear extracts from control, UV-B-stressed and wounded pea leaves. The pattern of DNA binding was almost identical with all three extracts, with one 30-bp region being the predominant site for factor binding. Using overlapping sub-fragments of this region, the majority of the specific binding could be attributed to the novel 11-bp GC-rich sequence GTGGCGCCCAC. An almost identical sequence is conserved in the sadA promoter. This motif has features in common with a number of recognised cis-elements, which suggests a possible binding site for factors which play a role in regulating sad gene transcription.

Suppression subtractive hybridisation was used to isolate genes differentially regulated by low levels (UV-B(BE,300) 0.13 W m(-2)) of ultraviolet-B radiation (UV-B; 290-320 nm) in Pisum sativum. Six genes were regulated, two of which were novel. The mRNA levels for these two (PsTSDC and PsUOS1) were increased and depressed by UV-B treatment, respectively. Domains in the PsTSDC translation product was similar to TIR (Toll-Interleukin-1 receptor-similar) domains and a NB-ARC domain (nucleotide-binding domain in APAF-1, R gene products and CED-4). The PsUOS1 translation product was similar to an open reading frame in Arabidopsis. Genes encoding embryo-abundant protein (PsEMB) and S-adenosyl-L-methionine synthase (PsSAMS) were induced by UV-B, whereas the transcript levels for genes encoding sucrose transport protein (PsSUT) or ribulose-5-phosphate 3-epimerase (PsR5P3E) were decreased. These regulation patterns are novel, and the PsEMB and PsR5P3E sequences are reported for the first time. The stress-specificity of regulation of these genes were tested by ozone fumigation (100 ppb O(3)). Qualitatively, the similarity of expression after both UV-B and ozone exposure suggests that, for these genes, similar stress-response pathways are in action.

A pea (Pisum sativum) DNA fragment (termed MB3) was isolated by differential display of cDNAs obtained from total leaf RNA of ultraviolet B (UV-B) radiation-treated plants. Longer cDNAs were cloned by rapid amplification of cDNA ends in the 3' to 5' direction. Three different, but very similar, cDNAs were cloned, sadA, sadB, and sadC, the major difference between them being a 36-bp deletion in the coding region of sadB. Southern blotting confirmed the occurrence of at least three genes in the pea genome. Database comparisons of the SAD protein sequences revealed high identity (46%) and similarity (77%) with a putative tomato (Lycopersicon esculentum) short-chain alcohol dehydrogenase. Very low levels of UV-B radiation (the biologically effective radiation normalized to 300 nm = 0.08 W m(-2)) was shown to up-regulate expression, a dose considerably lower than that needed to induce expression of the well-known UV-B defensive chalcone synthase and phenylalanine ammonia lyase genes. RNase protection assay revealed that primarily sadA and sadC mRNA accumulation was enhanced by UV-B. In addition to UV-B irradiation, ozone fumigation, wounding, aluminum stress, and salt stress induced increased transcript levels of the sad genes in pea.

The pra2 gene encodes a pea (Pisum sativum) small GTPase belonging to the YPT/rab family, and its expression is down-regulated by light, mediated by phytochrome. We have isolated and characterized a genomic clone of this gene and constructed a fusion DNA of its 5'-upstream region in front of the gene for firefly luciferase. Using this construct in a transient assay, we determined a pra2 cis-regulatory region sufficient to direct the light down-regulation of the luciferase reporter gene. Both 5'- and internal deletion analyses revealed that the 93-bp sequence between -734 and -642 from the transcriptional start site was important for phytochrome down-regulation. Gain-of-function analysis showed that this 93-bp region could confer light down-regulation when fused to the cauliflower mosaic virus 35S promoter. Furthermore, linker-scanning analysis showed that a 12-bp sequence within the 93-bp region mediated phytochrome down-regulation. Gel-retardation analysis showed the presence of a nuclear factor that was specifically bound to the 12-bp sequence in vitro. These results indicate that this element is a cis-regulatory element involved in phytochrome down-regulated expression.

As assayed by western blot analysis, red light induces the appearance of epitopes recognized by anti-phosphotyrosine antibodies in several pea nuclear proteins. The immunostaining is blocked by preadsorbing the antibodies with phosphotyrosine but not by preadsorbing them with phosphoserine or phosphothreonine. This light response is observed whether the red light irradiation is given to pea plumules or nuclei isolated from the plumules. The red-light-induced response seen in plumules is reversible by a subsequent far-red-light irradiation, indicating that the likely photoreceptor for this response may be phytochrome. By immunoblot analysis pea phytochrome A, but not phytochrome B, can be detected in proteins extracted from pea nuclear chromatin-matrix preparations. Phytochrome A and the protein bands immunostained by anti-phosphotyrosine antibodies can be solubilized from unirradiated pea chromatin by 0.3 M NaCl, but irradiating this preparation with red light does not induce the appearance of phosphotyrosine-like epitopes in any nuclear proteins. These results suggest that the association of phytochrome with purified pea nuclei is such that its conversion to the far-red light-absorbing form can induce a post-translational epitope change in nuclear proteins in vivo.

The blue-light-sensing apical part of coleoptiles of grasses is responsible for the first positive phototropic bending reaction of this organ. The photoreceptor responsible has been shown to be localized to the plasma membrane (PM) of this tip region. An approximately 100-kDa protein moiety of this receptor is rapidly phosphorylated upon irradiation. Properties of this protein kinase reaction were studied in vitro by using PMs from the maize (Zea mays L.) coleoptile tip region; (i) The substrate for the blue-light-triggered phosphorylation of the 100-kDa protein was found to be ATP as well as GTP. However, the affinity of the involved protein kinase for the substrate GTP was lower than for ATP. (ii) Experiments were undertaken to find out whether a photoreceptor moiety acts as an autophosphorylating protein kinase or whether the photoreceptor protein, when activated by light, becomes the target of an extrinsic protein kinase. Two studied extrinsic protein kinases (50 and 55 kDa) of the coleoptile tip were found not to be involved in the light-dependent protein phosphorylation. The degree of phosphorylation of the 100-kDa protein on isolated plasma membranes upon irradiation at 0 degrees C was scarcely different from a reaction at 30 degrees C, in contrast to the background protein phosphorylations which decreased with decreasing temperature. This result points to an autophosphorylation mechanism at the receptor. (iii) In mixing experiments, solubilized membranes from maize coleoptiles were irradiated and added to unirradiated membrane proteins from pea (Pisum sativum L.) epicotyls followed by addition of [gamma-32P]ATP. Unirradiated proteins from pea were not phosphorylated by light-activated (autophosphorylatable) maize protein kinases. (iv) It is suggested that the blue-light-sensitive photoreceptor localized to the PM of the phototropically active tip region of coleoptiles has an autophosphorylatable kinase domain which is able to use ATP or GTP as substrate.

Light activation of the pea (Pisum sativum) elip gene promoter was analysed in transgenic plants and in transiently transfected plant protoplasts. A series of promoter deletions fused to the gusA reporter was tested, and the results obtained by the two experimental approaches were in good agreement. We identified two nucleotide sequence elements involved in light-regulated expression of the elip gene. One element is similar to the GT1 binding site of the rbcS-3A gene, and the other resembles a G-box-like ACGT element. The region containing both elements was able to confer light responsiveness on a heterologous basic promoter. Electrophoretic mobility shift assays demonstrated that each element is specifically recognized by DNA-binding proteins present in nuclear extracts from pea seedlings. The G-box-like ACGT element is necessary but not sufficient for light inducibility, indicating that the two elements act together in confering light responsiveness.