Root Protein Extracts Research Articles

Toward alternative sources of milk coagulants for cheese manufacturing: establishment of hairy roots culture and protease characterization from Cynara cardunculus L.

Extracts from hairy root cultures of Cynara cardunculus L. contain proteases and show milk-clotting activity. Cynara cardunculus L. or cardoon is often used as rennet in traditional cheese manufacturing, due to the presence of specific proteases in the flower. However, the flower extracts are variable depending on the provenance and quality of the flowers as well as high genetic variability among cardoon populations, and this affects the quality of the final product. In search for alternative sources of milk-clotting enzymes, hairy root cultures from cardoon were obtained and characterized regarding their protease content and proteolytic activity toward milk proteins. Aspartic, serine and cysteine proteases were identified in hairy roots by mass spectrometry analysis and an azocasein assay combined with specific inhibitors. RT-PCR analysis revealed the expression of cardosin A and D, and immunoblotting analysis suggested the presence of cardosin A or cardosin A-like enzyme in its mature form, supporting this system as an alternative source of cardosins. Hairy root protein extracts showed activity over caseins, supporting its use as milk coagulant, which was further tested by milk-clotting assays. This is also the first report on the establishment of hairy root cultures from cardoon, which paves the way for future work on controlled platforms for production of valuable metabolites which are known to be present in this species.

Exogenous Auxin Elicits Changes in the Arabidopsis thaliana Root Proteome in a Time-Dependent Manner.

Auxin is involved in many aspects of root development and physiology, including the formation of lateral roots. Improving our understanding of how the auxin response is mediated at the protein level over time can aid in developing a more complete molecular framework of the process. This study evaluates the effects of exogenous auxin treatment on the Arabidopsis root proteome after exposure of young seedlings to auxin for 8, 12, and 24 h, a timeframe permitting the initiation and full maturation of individual lateral roots. Root protein extracts were processed to peptides, fractionated using off-line strong-cation exchange, and analyzed using ultra-performance liquid chromatography and data independent acquisition-based mass spectrometry. Protein abundances were then tabulated using label-free techniques and evaluated for significant changes. Approximately 2000 proteins were identified during the time course experiment, with the number of differences between the treated and control roots increasing over the 24 h time period, with more proteins found at higher abundance with exposure to auxin than at reduced abundance. Although the proteins identified and changing in levels at each time point represented similar biological processes, each time point represented a distinct snapshot of the response. Auxin coordinately regulates many physiological events in roots and does so by influencing the accumulation and loss of distinct proteins in a time-dependent manner. Data are available via ProteomeXchange with the identifier PXD001400.

Effects of exogenous auxin and ethylene on the Arabidopsis root proteome

The phytohormones, auxin and ethylene, together control a wide range of physiological and developmental processes in plants. The lack of knowledge regarding how the underlying signaling processes are reflected at the protein level represents a major gap in understanding phytohormone signaling, including that mediated by crosstalk between auxin and ethylene. Herein is a parallel comparison of the effects of these two hormones on the Arabidopsis root proteome. Arabidopsis seedlings were exposed to 1 μm indole-3-acetic acid (IAA, auxin) or 1 μm 1-amino-cyclopropane carboxylic acid (ACC) for 24h. Root protein extracts were fractionated using two-dimensional gel electrophoresis and the proteins that changed the most were analyzed by MALDI TOF/TOF mass spectrometry. Of the 500 total spots that were matched across all gels, 24 were significantly different after IAA exposure, while seven others were different after ACC exposure. Using rigorous criteria, identities of eight proteins regulated by IAA and five regulated by ACC were assigned. Interestingly, although both hormones affected proteins associated with fundamental cellular processes, no overlap was observed among the proteins affected by auxin or ethylene treatment. This report provides a comparison of the effects of these two hormones relative to a control utilizing equivalent treatment regimes and suggests that, while these hormones communicate to control similar physiological and transcriptional processes, they have different effects on the most abundant proteins in Arabidopsis roots.

Overexpression of OsPAP10a, A Root‐Associated Acid Phosphatase, Increased Extracellular Organic Phosphorus Utilization in Rice

Phosphorus (P) deficiency is a major limitation for plant growth and development. Among the wide set of responses to cope with low soil P, plants increase their level of intracellular and secreted acid phosphatases (APases), which helps to catalyze inorganic phosphate (Pi) hydrolysis from organo-phosphates. In this study we characterized the rice (Oryza sativa) purple acid phosphatase 10a (OsPAP10a). OsPAP10a belongs to group Ia of purple acid phosphatases (PAPs), and clusters with the principal secreted PAPs in a variety of plant species including Arabidopsis. The transcript abundance of OsPAP10a is specifically induced by Pi deficiency and is controlled by OsPHR2, the central transcription factor controlling Pi homeostasis. In gel activity assays of root and shoot protein extracts, it was revealed that OsPAP10a is a major acid phosphatase isoform induced by Pi starvation. Constitutive overexpression of OsPAP10a results in a significant increase of phosphatase activity in both shoot and root protein extracts. In vivo root 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) assays and activity measurements on external media showed that OsPAP10a is a root-associated APase. Furthermore, overexpression of OsPAP10a significantly improved ATP hydrolysis and utilization compared with wild type plants. These results indicate that OsPAP10a can potentially be used for crop breeding to improve the efficiency of P use.

Photosynthesis in aquatic adventitious roots of the halophytic stem‐succulent Tecticornia pergranulata (formerly Halosarcia pergranulata)

In flood-tolerant species, a common response to inundation is growth of adventitious roots into the water column. The capacity for these roots to become photosynthetically active has received scant attention. The experiments presented here show the aquatic adventitious roots of the flood-tolerant, halophytic stem-succulent, Tecticornia pergranulata (subfamily Salicornioideae, Chenopodiaceae) are photosynthetic and quantify for the first time the photosynthetic capacity of aquatic roots for a terrestrial species. Fluorescence microscopy was used to determine the presence of chloroplasts within cells of aquatic roots. Net O(2) production by excised aquatic roots, when underwater, was measured with varying light and CO(2) regimes; the apparent maximum capacity (P(max)) for underwater net photosynthesis in aquatic roots was 0.45 micromol O(2) m(-2) s(-1). The photosynthetic potential of these roots was supported by the immunolocalization of PsbA, the major protein of photosystem II, and ribulose-1-5-bisphosphate carboxylase/oxygenase (Rubisco) in root protein extracts. Chlorophyllous aquatic roots of T. pergranulata are photosynthetically active, and such activity is a previously unrecognized source of O(2), and potentially carbohydrates, in flooded and submerged plants.

Effects of cadmium and copper on peroxidase, NADH oxidase and IAA oxidase activities in cell wall, soluble and microsomal membrane fractions of pea roots

Twelve-day-old seedlings of pea ( Pisum sativum L.) that were treated for 4 days by 20 and 100 μmol/l Cd(NO 3) 2 or CuSO 4 showed a growth reduction in all organs. From root protein extracts, the activities of guaiacol peroxidase (GPX; EC 1.11.1.7), ascorbate peroxidase (APX; EC 1.11.1.11), coniferyl alcohol peroxidase (CAPX), NADH oxidase, and indole-3-acetic acid (IAA) oxidase were measured in covalently—and ionically—bound cell wall, soluble, and microsomal membrane fractions. With the exception of 20 μmol/l Cu, metal treatments enhanced GPX activity in all fractions. Only IAA oxidase activity was metal-elevated in the covalently bound cell wall fraction, while the ionic one showed Cd stimulation for all assayed enzymic activities. These effects were not entirely observed in Cu-treated plants, since APX and IAA oxidase activities were only enhanced in this fraction. However, soluble extract showed stimulation of APX activity, while in the microsomal fraction metal exposure also increased the activities of CAPX and NADH oxidase. Differential responses of root cell fractions to the presence of cadmium and copper ions are discussed in regard to the contribution of their enzymic capacities in antioxidant, lignification, and auxin degradation pathways. Comparisons between metals and dose effects are also underlined.

Proteome‐level changes in the roots of Pisum sativum in response to salinity

SummaryWe initiated a proteomics‐based approach to identify root proteins affected by salinity in pea (Pisum sativum cv. Cutlass). Salinity stress was imposed either on 2‐wk old pea plants by watering with salt water over 6 wk or by germinating and growing pea seeds for 7 days in Petri dishes. Concentrations of NaCl above 75 mM had significant negative effects on growth and development of peas in both systems. Salinity‐induced root proteome‐level changes in pea were investigated by 2‐D electrophoresis of proteins from control, 75 and 150 mM NaCl‐treated plants and seedlings. The majority of the protein spots visualised showed reproducible abundance in root protein extracts from whole plants and seedlings. Of these proteins, 35 spots that exhibited significant changes in abundance due to NaCl treatment were selected for identification using ESI‐Q‐TOF MS/MS. The identities of these proteins, which include pathogenesis‐related (PR) 10 proteins, antioxidant enzymes such as superoxide dismutase (SOD) as well as nucleoside diphosphate kinase (NDPK) are presented, and the roles of some of them in mediating responses of pea to salinity are discussed. This is the first report of salinity‐induced changes in the root proteome of pea that suggests a potential role for PR10 proteins in salinity stress responses. Our findings also suggest the possible existence of a novel signal transduction pathway involving SOD, H2O2, NDPK and PR10 proteins with a potentially crucial role in abiotic stress responses.

Structure-dependent phytotoxicity of catechins and other flavonoids: flavonoid conversions by cell-free protein extracts of Centaurea maculosa (spotted knapweed) roots.

Invasive plants are believed to succeed in part by secretion of allelochemicals, thus displacing competing plant species. Centaurea maculosa (spotted knapweed) provides a classic example of this process. We have previously reported that spotted knapweed roots secrete (+/-)-catechin and that (-)-catechin, but not (+)-catechin, is phytotoxic and hence may be a major contributor to C. maculosa's invasive behavior in the rhizosphere. In this communication, we explore both structure/activity relationships for flavonoid phytotoxicity and possible biosynthetic pathways for root production of (+/-)-catechin. Kaempferol and dihydroquercetin were shown to be phytotoxic, while quercetin was not. Kaempferol was converted to dihydroquercetin and (+/-)-catechin when treated with total root protein extracts from C. maculosa, but quercetin was not. This finding suggests an alteration in the standard flavonoid biosynthetic pathway in C. maculosa roots, whereby kaempferol is not a dead-end product but serves as a precursor to dihydroquercetin, which in turn leads to (+/-)-catechin production.

Localized versus systemic effect of arbuscular mycorrhizal fungi on defence responses to Phytophthora infection in tomato plants.

Development of biological control for plant diseases is accepted as a durable and environmentally friendly alternative for agrochemicals. Arbuscular mycorrhizal fungi (AMF), which form symbiotic associations with root systems of most agricultural, horticultural and hardwood crop species, have been suggested as widespread potential bioprotective agents. In the present study the ability of two AMF (Glomus mosseae and Glomus intraradices) to induce local or systemic resistance to Phytophthora parasitica in tomato roots have been compared using a split root experimental system. Glomus mosseae was effective in reducing disease symptoms produced by P. parasitica infection, and evidence points to a combination of local and systemic mechanisms being responsible for this bioprotector effect. The biochemical analysis of different plant defence-related enzymes showed a local induction of mycorrhiza-related new isoforms of the hydrolytic enzymes chitinase, chitosanase and beta-1,3-glucanase, as well as superoxide dismutase, an enzyme which is involved in cell protection against oxidative stress. Systemic alterations of the activity of some of the constitutive isoforms were also observed in non-mycorrhizal roots of mycorrhizal plants. Studies on the lytic activity against Phytophthora cell wall of root protein extracts also corroborated a systemic effect of mycorrhizal symbiosis on tomato resistance to Phytophthora.

Citrus rootstock responses to herbivory by larvae of the sugarcane rootstock borer weevil (Diaprepes abbreviatus)

The responses of roots to feeding by larvae of a citrus root weevil (Diaprepes abbreviatus) were investigated in Citrus grandis (L.) Osb. x Poncirus trifoliata (2N) (L.) Raf.; C. grandis x P. trifoliata (4N); P. trifoliata x C. grandis (Flying Dragon x Nakon); C. paradisi Macf. x P. trifoliata (Swingle citrumelo); C. aurantifolia (Christm.) Swingle (Citrus macrophylla); C. reticulata Blanco (Cleopatra mandarin); C. sinensis (L.) Osb. x P. trifoliata (Carrizo citrange); C. aurantium (L.) (sour orange). Chitinase, chitosanase. β‐1,3‐glucanase, peroxidase and lysozyme activities were measured and significant differences were observed for some of the cultivars between infested and uninfested rootstocks. Generally, increased activities were observed for chitinases and decreased activities were observed for the other enzymes measured. Numerous significant differences in hydrolase and peroxidase activities were observed between cultivars. Immunological detection revealed that new protein bands occurred in root protein extracts for six of the eight cultivars infested with larvae when an antibody to a class I potato leaf chitinase was used. Antibodies generated against two citrus chitinases of Mr 24 000 (basic chitinase cv. Valencia (C. sinensis) callus, BCVC) and Mr 28 000 (basic chitinase/lysozyme cv. Valencia callus, BCLVC) indicated that chitinases in Carrizo were induced in infested roots when the BCVC antibody was employed. These findings justify calling these proteins pathogenesis‐related proteins. The chitinase that BCLVC was prepared from exhibited high lysozyme activities, and the results of western blots showed the presence of proteins at Mr 24 000 and 27 000 which are presumed to be lysozymes. Similar tests using antibodies against β‐1, 3‐glucanases and peroxidases indicated a diminution of protein bands that cross‐reacted with infested root protein extracts compared with what occurred in controls. All of the root extracts were tested against chitosans with various percentages of acetylation; activities were linearly dependent on the amount of chitosan acetylation; i.e. the larger the amount of acetylation, the greater the activity. Significant differences in hydrolase activities were observed between infested and uninfested roots for the rootstocks using the variously acetylated substrates. All of the root protein extracts were capable of degrading peritrophic membranes removed from larvae of D. abbreviatus. This suggests that citrus chitinases may play a role in disrupting the peritrophic membrane such that ingested substances that pose a hazard to the insect may penetrate the membrane more easily.

The glutamine synthetase gene family of Arabidopsis thaliana light-regulation and differential expression in leaves, roots and seeds

Glutamine synthetase (GS) plays an important role in the assimilation of nitrogen by higher plants. We present here a molecular analysis of the GS polypeptides, mRNAs, and genes of Arabidopsis thaliana. Western blot analysis of leaf and root protein extracts revealed at least two distinct GS polypeptides; 43 kDa and 39 kDa GS polypeptides were present in leaves, while only a 39 kDa GS was detected in roots. The 43 kDa GS polypeptide is light-inducible. In etiolated seedlings only the 39 kDa GS was detected. However, upon greening the 43 kDa GS increased to levels comparable to those observed in light-grown plants. Four distinct GS cDNA clones, lambda Atgsl1, lambda Atgsr1, lambda Atgsr2 and lambda Atkb6 were isolated and characterized. Their complete nucleotide and deduced amino acid sequences are presented. The coding sequences of the four clones are 70-88% similar while their 5' and 3' untranslated regions exhibit less than 50% similarity. Northern blots of leaf, root and germinated seed RNA revealed that the four cDNAs hybridize to mRNAs which are differentially expressed in the organs of Arabidopsis thaliana. lambda Atgsl1 is leaf-specific and hybridizes to a 1.6 kb mRNA. Both lambda Atgsr1 and lambda Atgskb6 hybridize to 1.4 kb mRNAs which are expressed in both roots and germinated seeds. lambda Atgsr2 hybridizes to a 1.4 kb mRNA, which is primarily expressed in roots with low levels of expression in seeds and leaves. lambda Atgsl1, which represents the leaf-specific mRNA, is induced by light. lambda Atgsl1 mRNA levels increase during the greening of etiolated seedlings while lambda Atgsr1 levels remain constant. Southern blot analysis indicated that the Arabidopsis genome contains at least four and possibly five distinct GS genes.

A Homolog of the Substrate Adhesion Molecule Vitronectin Occurs in Four Species of Flowering Plants.

The extracellular matrix (ECM) has been implicated in the primary developmental processes of many organisms. A family of secretory adhesive glycoproteins called substrate adhesion molecules (SAMs) is believed to confer these dynamic capabilities to the ECM in animals. In this paper, we report the existence of SAM-like genes and gene products in flowering plants. Hybridizations with a human vitronectin cDNA probe and genomic DNA from broad bean, soybean, and tomato revealed vitronectin-like sequences. Human vitronectin antibodies cross-react with a 55-kilodalton protein in leaf and root protein extracts from lily, broad bean, soybean, and tomato. In addition, immunocytochemical staining of frozen sections of lily leaf and broad bean gynoecium demonstrated that vitronectin-like proteins were localized to the ECM on the cell surface, with the most intense labeling residing in the transmitting tract of broad bean gynoecium.

Ferric Reducing Activity in Roots of Fe-deficient Phaseolus vulgaris Source of Reducing Equivalents

Summary Fe deficiency activates a system in the roots of dicotyledons which can reduce extracellular ferric chelates (ferric reductase); this system is located in the plasma membranes of the epidermal cells. In order to identify the source of reducing equivalents for the reduction of ferric chelates by roots of bean ( Phaseolus vulgaris L. var. Prelude), properties of NADP + reducing enzymes were studied and incubations were performed with glucose labeled with 14 C at positions Cl and C6. NADP-dependent isocitrate dehydrogenase was the most active of the enzymes tested in root protein extracts and it could reduce NADP+ even at very high NADPH/NADP + ratios. It is concluded that citrate, which accumulates in roots upon Fe deficiency, via transformation into isocitrate by mitochondrial aconitase, drives cytosolic NADP in bean roots to a strongly reduced state through isocitrate dehydrogenase. Upon addition of reducible ferric salts, the NADP couple becomes more oxidized making it possible for the oxidative pentose phosphate pathway to contribute to NADP + reduction.