Non-photosynthetic Plant Tissues Research Articles

Oxidative Stress and Marine Carotenoids: Application by Using Nanoformulations.

Carotenoids are natural fat-soluble pigments synthesized by plants, algae, fungi and microorganisms. They are responsible for the coloration of different photosynthetic organisms. Although they play a role in photosynthesis, they are also present in non-photosynthetic plant tissues, fungi, and bacteria. These metabolites have mainly been used in food, cosmetics, and the pharmaceutical industry. In addition to their utilization as pigmentation, they have significant therapeutically applications, such as improving immune system and preventing neurodegenerative diseases. Primarily, they have attracted attention due to their antioxidant activity. Several statistical investigations indicated an association between the use of carotenoids in diets and a decreased incidence of cancer types, suggesting the antioxidant properties of these compounds as an important factor in the scope of the studies against oxidative stress. Unusual marine environments are associated with a great chemical diversity, resulting in novel bioactive molecules. Thus, marine organisms may represent an important source of novel biologically active substances for the development of therapeutics. Marine carotenoids (astaxanthin, fucoxanthin, β-carotene, lutein but also the rare siphonaxanthin, sioxanthin, and myxol) have recently shown antioxidant properties in reducing oxidative stress markers. Numerous of bioactive compounds such as marine carotenoids have low stability, are poorly absorbed, and own very limited bioavailability. The new technique is nanoencapsulation, which can be used to preserve marine carotenoids and their original properties during processing, storage, improve their physiochemical properties and increase their health-promoting effects. This review aims to describe the role of marine carotenoids, their potential applications and different types of advanced nanoformulations preventing and treating oxidative stress related disorders.

Leucoplast Isolation and Subfractionation.

Leucoplasts are colorless plastids of nonphotosynthetic plant tissues that support a variety of anabolic roles, particularly the biosynthesis of long-chain fatty acids in storage tissues of developing oil seeds. They also perform other important metabolic functions including the biosynthesis of amino acids and tetrapyrrole compounds. Leucoplasts use a complex set of membrane carriers and channels to actively translocate nuclear-encoded precursor proteins from the cytosol, while exchanging various metabolites with the cytosol. Leucoplast purification is a necessary prerequisite for detailed studies of their soluble (stromal) and membrane (envelope) (phospho)proteomes, as well as for achieving a detailed understanding of their metabolic capabilities, transport processes, and biogenesis. This chapter describes protocols for leucoplast purification from endosperm of developing castor oil seeds, and their subsequent subfractionation into envelope membrane and soluble stromal compartments for biochemical analysis.

Synthesis and Use of Stable-Isotope-Labeled Internal Standards for Quantification of Phosphorylated Metabolites by LC–MS/MS

Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is a highly specific and sensitive technique for measuring metabolites. However, coeluting components in tissue extracts can interfere with ionization at the interface of the LC and MS/MS phases, causing under- or overestimation of metabolite concentrations. Spiking of samples with known amounts of stable-isotope-labeled internal standards (SIL-IS) allows measurements of the corresponding metabolites to be corrected for such matrix effects. We describe criteria for selection of suitable SIL-IS and report the enzymatic synthesis and purification of nine SIL-IS for hexose-, pentose-, and triose-phosphates, UDP-glucose, and adenosine monophosphate (AMP). Along with commercially available SIL-IS for seven other metabolites, these were validated by LC-MS/MS analyses of extracts from leaves, nonphotosynthetic plant tissues, mouse liver, and cells of Chlamydomonas reinhardtii, Escherichia coli and baker's yeast (Saccharomyces cerevisiae). With only a few exceptions, spiking with SIL-IS significantly improved the reproducibility of LC-MS/MS-based metabolite measurements across a wide range of extract dilutions, indicating effective correction for matrix effects by this approach. With use of SIL-IS to correct for matrix effects, LC-MS/MS offers unprecedented scope for reliable determination of photosynthetic and respiratory intermediates in a diverse range of organisms.

Chloroplast redox homeostasis is essential for lateral root formation in Arabidopsis

Redox regulation based on dithiol-disulphide interchange is an essential component of the control of chloroplast metabolism. In contrast to heterotrophic organisms, and non-photosynthetic plant tissues, chloroplast redox regulation relies on ferredoxin (Fd) reduced by the photosynthetic electron transport chain, thus being highly dependent on light. The finding of the NADPH-dependent thioredoxin reductase C (NTRC), a chloroplast-localized NTR with a joint thioredoxin domain, showed that NADPH is also used as source of reducing power for chloroplast redox homeostasis. Recently we have found that NTRC is also in plastids of non-photosynthetic tissues. Because these non-green plastids lack photochemical reactions, their redox homeostasis depends exclusively on NADPH produced from sugars and, thus, NTRC may play an essential role maintaining the redox homeostasis in these plastids. The fact that redox regulation occurs in any type of plastids raises the possibility that the functions of chloroplasts and non-green plastids, such as amyloplasts, are integrated to harmonize the growth of the different organs of the plant. To address this question, we generated Arabidopsis plants the redox homeostasis of which is recovered exclusively in chloroplasts, by leaf-specific expression of NTRC in the ntrc mutant, or exclusively in amyloplasts, by root-specific expression of NTRC. The analysis of these plants suggests that chloroplasts exert a pivotal role on plant growth, as expected because chloroplasts constitute the major source of nutrients and energy, derived from photosynthesis, for growth of heterotrophic tissues. However, NTRC deficiency causes impairment of auxin synthesis and lateral root formation. Interestingly, recovery of redox homeostasis of chloroplasts, but not of amyloplasts, was sufficient to restore wild type levels of lateral roots, showing the important signaling function of chloroplasts for the development of heterotrophic organs.

NADPH Thioredoxin Reductase C Is Localized in Plastids of Photosynthetic and Nonphotosynthetic Tissues and Is Involved in Lateral Root Formation in Arabidopsis

Plastids are organelles present in photosynthetic and nonphotosynthetic plant tissues. While it is well known that thioredoxin-dependent redox regulation is essential for leaf chloroplast function, little is known of the redox regulation in plastids of nonphotosynthetic tissues, which cannot use light as a direct source of reducing power. Thus, the question remains whether redox regulation operates in nonphotosynthetic plastid function and how it is integrated with chloroplasts for plant growth. Here, we show that NADPH-thioredoxin reductase C (NTRC), previously reported as exclusive to green tissues, is also expressed in nonphotosynthetic tissues of Arabidopsis thaliana, where it is localized to plastids. Moreover, we show that NTRC is involved in maintaining the redox homeostasis of plastids also in nonphotosynthetic organs. To test the relationship between plastids of photosynthetic and nonphotosynthetic tissues, transgenic plants were obtained with redox homeostasis restituted exclusively in leaves or in roots, through the expression of NTRC under the control of organ-specific promoters in the ntrc mutant. Our results show that fully functional root amyloplasts are not sufficient for root, or leaf, growth, but fully functional chloroplasts are necessary and sufficient to support wild-type rates of root growth and lateral root formation.

Summer water use by California coastal prairie grasses: fog, drought, and community composition

Plants in the Mediterranean climate region of California typically experience summer drought conditions, but correlations between zones of frequent coastal fog inundation and certain species' distributions suggest that water inputs from fog may influence species composition in coastal habitats. We sampled the stable H and O isotope ratios of water in non-photosynthetic plant tissue from a variety of perennial grass species and soil in four sites in northern California in order to determine the proportion of water deriving from winter rains and fog during the summer. The relationship between H and O stable isotopes from our sample sites fell to the right of the local meteoric water line (LMWL) during the summer drought, providing evidence that evaporation of water from the soil had taken place prior to the uptake of water by vegetation. We developed a novel method to infer the isotope values of water before it was subjected to evaporation in which we used experimental data to calculate the slope of the deltaH versus deltaO line versus the LMWL. After accounting for evaporation, we then used a two-source mixing model to evaluate plant usage of fog water. The model indicated that 28-66% of the water taken up by plants via roots during the summer drought came from fog rather than residual soil water from winter rain. Fog use decreased as distance from the coast increased, and there were significant differences among species in the use of fog. Rather than consistent differences in fog use by species whose distributions are limited to the coast versus those with broader distributions, species responded individualistically to summer fog. We conclude that fogwater inputs can mitigate the summer drought in coastal California for many species, likely giving an advantage to species that can use it over species that cannot.

Overexpression of the enzyme p-hydroxyphenolpyruvate dioxygenase in Arabidopsis and its relation to tocopherol biosynthesis

The enzyme p-hydroxyphenylpyruvate dioxygenase (HPPD) catalyzes the conversion of p-hydroxyphenylpyruvate to homogentisic acid (HGA), the aromatic precursor for the biosynthesis of vitamin E (α-tocopherol) and plastoquinone. In order to determine if increased HPPD activity could positively impact tocopherol yields, transgenic plants were generated that overexpressed the gene encoding Arabidopsis HPPD. Transgenic plants exhibiting high levels of HPPD expression were identified by increased tolerance to a competitive inhibitor of HPPD, the herbicide sulcotrione. HPPD gene expression in these transgenic lines, as determined at the RNA, protein and activity levels, was at least 10-fold higher than that of wild-type plants. Subsequent tocopherol analysis of leaf and seed material revealed that the increased HPPD expression resulted in up to a 37% increase in leaf tocopherol levels and a 28% increase in seed tocopherol levels relative to control plants. These results demonstrate that HPPD activity, and likely HGA levels, are at least one factor limiting the production of tocopherols in photosynthetic and non-photosynthetic plant tissues.

Strategies for metabolic flux analysis in plants using isotope labelling

Flux measurements through metabolic pathways generate insights into the integration of metabolism, and there is increasing interest in using such measurements to quantify the metabolic effects of mutation and genetic manipulation. Isotope labelling provides a powerful approach for measuring metabolic fluxes, and it gives rise to several distinct methods based on either dynamic or steady-state experiments. We discuss the application of these methods to photosynthetic and non-photosynthetic plant tissues, and we illustrate the different approaches with an analysis of the pathways interconverting hexose phosphates and triose phosphates. The complicating effects of the pentose phosphate pathway and the problems arising from the extensive compartmentation of plant cell metabolism are considered. The non-trivial nature of the analysis is emphasised by reference to invalid deductions in earlier work. It is concluded that steady-state isotopic labelling experiments can provide important information on the fluxes through primary metabolism in plants, and that the combination of stable isotope labelling with detection by nuclear magnetic resonance is particularly informative.

Free radical formation in non-photosynthetic plant tissues — an overview

Free radical formation in non-photosynthetic plant tissues — an overview

Differentiation of Six Distinct Thioredoxins in Seeds of the Soybean

The thioredoxin profile of non-photosynthetic plant tissues, in particular of seeds, is much less known than that of green leaves. We have systematically fractionated and assayed the low molecular weight, heat-stable proteins present in extracts from soybean seeds by a variety of chromatographic and enzymatic methods. A multiplicity of six thioredoxins has been established, closely comparable to the pattern previously observed in soybean leaves (Häberlein,1991). Purification to homogeneity of the six proteins was achieved by FPLC. Thioredoxin activities in vitro towards soybean chloroplast NADP-MDH, fructosebisphosphatase, NADPH thioredoxin reductase, and E. coli ribonucleotide reductase have been measured. Some of the seed thioredoxins exhibit higher molecular weights, stronger binding to anion exchange columns, and much lower specificity in enzyme assays than their counterparts isolated from leaves, suggesting the presence of unspecialized storage forms in the dry seed.

Identification of a Major Soluble Protein in Mitochondria from Nonphotosynthetic Tissues as NAD-Dependent Formate Dehydrogenase

In many plant species, one of the most abundant soluble proteins (as judged by two-dimensional polyacrylamide gel electrophoresis) in mitochondria from nongreen tissues is a 40-kD polypeptide that is relatively scarce in mitochondria from photosynthetic tissues. cDNA sequences encoding this polypeptide were isolated from a lambda gt11 cDNA expression library from potato (Solanum tuberosum L.) by screening with a specific antibody raised against the 40-kD polypeptide. The cDNA sequence contains an open reading frame of 1137 nucleotides whose predicted amino acid sequence shows strong homology to an NAD-dependent formate dehydrogenase (EC 1.2.1.2) from Pseudomonas sp. 101. Comparison of the cDNA sequence with the N-terminal amino acid sequence of the mature 40-kD polypeptide suggests that the polypeptide is made as a precursor with a 23-amino acid presequence that shows characteristics typical of mitochondrial targeting signals. The identity of the polypeptide was confirmed by assaying the formate dehydrogenase activity in plant mitochondria from various tissues and by activity staining of mitochondrial proteins run on native gels combined with antibody recognition. The abundance and distribution of this protein suggest that higher plant mitochondria from various nonphotosynthetic plant tissues (tubers, storage roots, seeds, dark-grown shoots, cauliflower heads, and tissues grown in vitro) might contain a formate-producing fermentation pathway similar to those described in bacteria and algae.

Characterization of a ferredoxin:NADP + oxidoreductase from a nonphotosynthetic plant tissue

A flavoprotein with properties similar to those of ferredoxin:NADP + oxidoreductases found in the leaves of higher plants has been purified to apparent homogeneity from bean sprouts, a nonphotosynthetic plant tissue. The absorbance and circular dichroism spectra of the bean sprout protein are similar to those of spinach leaf ferredoxin:NADP + oxidoreductase and an antibody raised against the spinach enzyme recognized the bean sprout enzyme. The bean sprout enzyme catalyzed ferredoxin-dependent electron transfer from NADPH to equine cytochrome c at a high rate but, unlike the spinach enzyme, exhibited little NADPH to 2,6-dichlorophenol indophenol diaphorase activity. The bean sprout enzyme forms a 1:1 electrostatically stabilized complex with ferredoxins isolated from either bean sprouts or spinach leaves.

Cellular energy levels and their effect on male cell abortion in cytoplasmically male sterile lines of Petunia hybrida

Using an HPLC method it has proved possible to follow the levels of adenine and pyridine nucleotides in the anthers of normal and cytoplasmically male sterile (CMS) lines of Petunia hybrida. Well before the appearance of any structural differences, anthers of CMS plants begin to show lower ATP/ADP ratios. In anthers, as in other non-photosynthetic plant tissues, there is a strong correlation between the ATP/ADP ratio and levels of NADPH and, when NADPH was assayed in fertile and CMS anthers, very dramatic differences were discovered. In male fertile plants, the NADPH/NADP ratio differs strikingly between somatic and reproductive tissues and reaches a peak in anthers at the early prophase of meiosis. The ratio in male sterile anthers remains largely unchanged from that normally seen in somatic tissues over this period. A cytochemical localisation technique revealed that, at the stage of development in question, the major reserves of anther NADPH are held within the tapetal cells and that levels within CMS tapeta are strikingly lower than those of normal plants. These findings are discussed in the perspective of theories proposed to explain the operation of CMS, and also in terms of the now conclusive evidence that the genetical basis of CMS lies in changes in the organisation of the mitochondrial genome.

Evidence for the presence of a [2Fe-2S] ferredoxin in bean sprouts

An iron-sulfur protein with properties similar to those of ferredoxins found in the leaves of higher plants has been isolated from bean sprouts - a non-photosynthetic plant tissue. The bean sprout protein has a molecular mass of 12.5 kDa and appears to contain a single [2Fe-2S] cluster. The absorbance and circular dichroism spectra of the bean sprout protein resemble those of spinach leaf ferredoxin and the bean sprout protein can replace spinach ferredoxin as an electron donor for NADP + reduction, nitrite reduction and thioredoxin reduction by spinach leaf enzymes. Although the reduced bean sprout protein ( E m = −440 mV) is a slightly stronger reductant than spinach ferredoxin and appears to be less acidic than spinach ferredoxin, the two proteins are similar enough so that the bean sprout protein is recognized by an antibody raised against spinach ferredoxin.

Proline Accumulation in Tubers of Jerusalem Artichoke

When separated whole tubers of Helianthus tuberosus L. were dried, and when tuber slices were plasmolysed in mannitol solutions, free proline accumulated in the tuber tissue. The amount of proline accumulated in tuber slices increased with the concentration of mannitol in the plasmolysing solution. A lag of approximately 24 h preceded proline accumulation in plasmolysed slices. During the lag period, the content of free arginine, a major component of the free amino acid pool, decreased by at least 50%. L-Indospicine, a competitive inhibitor of arginase (L-arginine amidinohydrolase EC 3.5.3.1) inhibited arginine catabolism in slices and also inhibited proline accumulation. On the other hand, cycloheximide, an inhibitor of protein synthesis, did not inhibit arginine loss or proline accumulation. An increase in the concentration of mannitol required for plasmolysis accompanied an increase in free proline in whole, dormant tubers stored at 4°C. These tubers are an example of a non-photosynthetic higher plant tissue in which free proline accumulates when the water potential decreases. The relationship between arginine catabolism and proline accumulation is discussed.

The Presence of Ribulose 1,5-Diphosphate Carboxylase in the Nonphotosynthetic Endosperm of Germinating Castor Beans

Ribulose 1,5-diphosphate carboxylase was detected in extracts of germinating castor bean (Ricinus communis var. Hale) endosperms. This is the first report of this enzyme in a nonphotosynthetic (no chlorophyll) plant tissue. Radioactive 3-phosphoglyceric acid has been identified as the principle product resulting from the enzymatic condensation of (14)C-bicarbonate and ribulose-1,5-diP in endosperm extracts. The Km values of bicarbonate and ribulose-1,5-diP for the endosperm carboxylase are 1.14 x 10(-2)m and 7.5 x 10(-5)m, respectively. The carboxylase activity peaks at 4 days in endosperms of castor beans germinated in the dark. The specific activity of the carboxylase at this stage of germination is 4.3 mumoles of 3-phosphoglycerate formed/mg protein.hr. The presence of ribulose-1,5-diP carboxylase and other enzymes of the reductive pentose phosphate pathway show the potential of this pathway in castor bean endosperms.

Compartmentation of nicotinamide dinucleotide dehydrogenases and transhydrogenases in nonphotosynthetic plant tissues

The intracellular distributions of a number of NAD +-specific and NADP +-specific dehydrogenases in the stems of etiolated pea seedlings and in other nonphotosynthetic plant tissues have been determined. The NADP +-specific enzymes were found to be located almost exclusively in the soluble fraction, whereas the NAD +-specific enzymes were predominantly mitochondrial. There was no appreciable NADPH → NAD + transhydrogenase activity in any of the isolated fractions. On the other hand, active homotranshydrogenases of the type NADH → NAD + and NADPH → NADP + were detected; the former is predominantly mitochondrial, while the latter is found in both the microsomal and soluble fractions. The possibility that these transhydrogenase activities are due to enzymes which normally catalyze other reactions is considered. The results support the view that in plant tissues, as in animals, there is a “division of labor” between NAD and NADP.

Oxidation of Menadiol by Fractions Isolated From Non-Photosynthetic Plant Tissues

The role of quinones in electron transport has attracted considerable recent interest (27). Coenzyme Q1,O which is present in plant mitochondria, is closely related to the respiratory chain (6), and the very similar benzoquinone, plastoquinone, appears to be involved in photosynthesis (7); Vitamin K-type naphthoquinones have been implicated in respiratory electron transfer (30), oxidative phosphorylation (2), and photophosphorylation (1). The enzymatic pathways involved in the intracellular oxidation and reduction of these quinones are clearly of interest. Wosilait and Nason (32) showed that extracts of animals, plants, and bacteria contain an active pyridine nucleotide-menadione (vitamin K3) reductase, and since then a number of enzymes which catalyze the reduction of naphthoquinones and benzoquinones or benzoquinones alone have been purified. The pathways of oxidation of the reduced quinones have also been examined, though most of the work has been done with animal systems (8,9,22). This aspect of the problem is of special interest in plants, since, in addition to the usual respiratory chain, they contain a number of enzymes (phenol oxidase, laccase, peroxidase) which can oxidize phenols to their corresponding quinones. In the present study, we have used menadiol and menadione as model compounds and have examined their oxidation and reduction by cell-free fractions isolated from non-photosynthetic tissues. Special attention has been paid to the intracellular localization of these activities and to the components involved in the oxidative pathways.

Triphosphopyridine Nucleotide-linked Glyceraldehyde Phosphate Dehydrogenase in a Non-Photosynthetic Plant Tissue

IT has been established that, whereas the usual diphosphopyridine nucleotide (DPN)-linked glyceraldehyde-phosphate dehydrogenase is found generally distributed in higher plant tissues, a separate enzyme, specifically requiring triphosphopyridine nucleotide (TPN) as coenzyme, is restricted to leaves1–4. The distinctive association of the TPN-glyceraldehyde phosphate dehydrogenase with the photosynthetic apparatus and the specificity for TPN shown in some types of photosynthetic phosphorylation have encouraged the view that the reductive step in hexose synthesis in leaves is mediated by this enzyme.

Haem pigments of cytoplasmic particles from nonphotosynthetic plant tissues.

Haem pigments of cytoplasmic particles from nonphotosynthetic plant tissues.