Metabolism of the folate precursor p-aminobenzoate in plants: glucose ester formation and vacuolar storage

Plants produce p-aminobenzoate (pABA) in chloroplasts and use it for folate synthesis in mitochondria. In plant tissues, however, pABA is known to occur predominantly as its glucose ester (pABA-Glc), and the role of this metabolite in folate synthesis has not been defined. In this study, the UDP-glucose:pABA acyl-glucosyltransferase (pAGT) activity in Arabidopsis extracts was found to reside principally (95%) in one isoform with an apparent K(m) for pABA of 0.12 mm. Screening of recombinant Arabidopsis UDP-glycosyltransferases identified only three that recognized pABA. One of these (UGT75B1) exhibited a far higher k(cat)/K(m) value than the others and a far lower apparent K(m) for pABA (0.12 mm), suggesting its identity with the principal enzyme in vivo. Supporting this possibility, ablation of UGT75B1 reduced extractable pAGT activity by 95%, in vivo [(14)C]pABA glucosylation by 77%, and the endogenous pABA-Glc/pABA ratio by 9-fold. The K(eq) for the pABA esterification reaction was found to be 3 x 10(-3). Taken with literature data on the cytosolic location of pAGT activity and on cytosolic UDP-glucose/UDP ratios, this K(eq) value allowed estimation that only 4% of cytosolic pABA is esterified. That pABA-Glc predominates in planta therefore implies that it is sequestered away from the cytosol and, consistent with this possibility, vacuoles isolated from [(14)C]pABA-fed pea leaves were estimated to contain> or =88% of the [(14)C]pABA-Glc formed. In total, these data and the fact that isolated mitochondria did not take up [(3)H]pABA-Glc, suggest that the glucose ester represents a storage form of pABA that does not contribute directly to folate synthesis.     

Lipid composition of plant mitochondria and of chloroplasts

The mitochondrial lipids from avocado fruit, cauliflower buds, and potato tubers, and the lipids of chloroplasts isolated from avocado fruit and from cauliflower leaves were identified and the concentrations were determined. The lipid composition was compared with that of beef heart mitochondria. Phospholipids constituted 50-56% of total lipids in plant mitochondria while this fraction made up 90% of the lipids in beef heart mitochondria. In both cases the chief phospholipids were phosphatidylcholine and phosphatidylethanolamine. A characteristic feature of plant mitochondria was the presence of monogalactosyl- and digalactosyldiglyceride and of sulfolipid. Potato mitochondria differed from the particles of other species investigated by their higher content of galactolipids, sterol glycosides, and carotenoids and lower content of phospholipids and of total lipids in the lipidprotein complex. The galactolipid content was markedly higher in chloroplasts from all sources than in mitochondria. The spectrum of lipids in the phospholipid fraction differed more strikingly between chloroplasts of the leaf and the mitochondria of the bud of cauliflower than between the two organelles of the avocado mesocarp. The fatty acid distribution of individual lipids and of classes of lipids was also more similar in the two organelles of the fruit tissue than in the cauliflower material.     

Hydrolysis of dolichyl esters by rat liver lysosomes

Dolichyl ester hydrolase activity is broadly distributed among the organs of the rat. The highest activity was found in spleen, brain, lung, and thyroid tissues, whereas this activity is very low in stomach and intestine. The esterase involved is localized to the lumen of lysosomes and, to some extent, in the plasma membranes. Hydrolysis occurs with both alpha-saturated and alpha-unsaturated polyisoprenes esterified with different fatty acids, but the rate of hydrolysis is strongly dependent on the nature of the substrate. The enzyme involved is inhibited by divalent cations, EDTA and EGTA and also by one of the products, dolichol. The esterase is activated by 3-[(3-cholamidopropyl) dimethylammonio]-1-propranesulfonic acid and taurodeoxycholate and inhibited by Triton X-100. Dolichyl esterase activity is completely inhibited by alpha- and beta-naphthyl acetate, phenylmethylsulfonyl fluoride, and beta-chloromethylmercurisulfate. These inhibitors, as well as the pH optimum for dolichyl ester hydrolysis, clearly differentiate the enzyme involved from cholesteryl esterase and triglyceride lipase. Microsomal phospholipase A hydrolyzes dolichyl esters at a slow rate only. In vivo labeling experiments with [3H]mevalonate demonstrated that newly synthesized dolichol is transported in esterified form to the lysosomes, where this lipid is slowly hydrolyzed by the esterase. The possibility is raised that the role of the fatty acyl moiety may be to target dolichol to its final location in the cell.     

Subcellular distribution, multiple forms and development of glutamate-pyruvate (glyoxylate) aminotransferase in plant tissues

According to a sucrose density gradient analysis of cell organelles from homogenates of green leaves of rye, wheat and pea seedlings glutamate-pyruvate aminotransferase was predominantly localized in the leaf microbodies (peroxisomes; 90%) and to a minor extent in the mitochondria (10%) but completely absent from chloroplasts. In etiolated rye leaves the distribution of the enzyme was similar. In other non-green tissues glutamate-pyruvate aminotransferase was predominantly associated with the mitochondria but also present in the microbodies of dark-grown pea roots and in the glyoxysomes of Ricinus endosperm. In the microbodies isolated from potato tubers the enzyme was not detectable. Glutamate-pyruvate aminotransferase activity was not associated with the proplastid fractions of the non-green tissues. The distribution of glutamate-oxaloacetate aminotransferase was different from that of glutamate-pyruvate aminotransferase. Glutamate-oxaloacetate aminotransferase was found in chloroplasts, proplastids, mitochondria, microbodies and in the supernatant. Evidence is presented that glutamate-pyruvate and glutamate-glyoxylate aminotransferase activities were catalyzed by the same enzyme. Both activities showed the same organelle distribution on sucrose gradients and both were eluted at the same salt concentration from DEAE-cellulose. By chromatography of preparations from rye leaf extracts on DEAE-cellulose two forms of glutamate-pyruvate (glyoxylate) aminotransferase were separated. The major fraction eluting at a low salt concentration was identified as peroxisomal form and the minor fraction eluting at a higher salt concentration was identified as a mitochondrial form. Both the glutamate-glyoxylate and the glutamate-pyruvate aminotransferase activities of the peroxisomal as well as of the mitochondrial forms of the enzyme were strongly (about 80%) inhibited by the presence of 10 mM glycidate, previously described as an inhibitor of glutamate-glyoxylate aminotransferase in tobacco tissue. Pig heart glutamate-pyruvate aminotransferase exhibited no glutamate-glyoxylate aminotransferase activity and was only slightly inhibited by glycidate. The development of glutamate-pyruvate aminotransferase activity in the leaves of rye seedlings was strongly increased in the light, relative to dark-grown seedlings, and very similar to that of catalase activity while the development of glutamate-oxaloacetate aminotransferase was, in close coincidence with the behavior of leaf growth, only slightly enhanced by light. It is discussed that in green leaves an extrachloroplastic synthesis of alanine is of considerable advantage for the metabolic flow during photosynthesis.     

Intracellular site of sucrose synthesis in leaves

Improved conditions for extraction and assay increased rates of sucrose synthesis from uridine diphosphate glucose (UDPglucose) plus fructose 6-phosphate (F.6.P) catalysed by leaf extracts 20-fold. Rates of 17.9, 25·0, 9·2 and 27·7 μmol/hr/g fr. wt respectively were obtained from pea shoots, spinach, wheat and bean leaves. Chloroplasts isolated from pea shoots, in which half the plastids were intact, contained less than 4% of the total UDPglucose-fructosephosphate glucosyltransferase, more than 30% of the ribulose diphosphate (RuDP) carboxylase, and more than 40% of the total chlorophyll of the leaf. Although some of the UDPglucose-fructose-phosphate glucosyltransferase was associated with particles smaller than chloroplasts at least 85% of the enzyme was not precipitated at 38 000 g. UDPglucose pyrophosphorylase, also thought to be essential for sucrose synthesis, was distributed between the cell fractions in a similar manner to UDPglucose-fructosephosphate glucosyltransferase. It is concluded that sucrose synthesis in pea shoots and spinach leaves occurs mainly, in the cytoplasm.     

Reconstitution of amino acid synthesis by combining spinach chloroplasts with other leaf organelles

By adding leaf peroxisomes to purified intact chloroplasts, glycine synthesis was reconstituted. On adding leaf mitochondria, serine synthesis was also reconstituted. However, aromatic amino acid synthesis which was effected by purified chloroplasts was not enhanced on adding peroxisomes or mitochondria although the rate in whole leaves was considerably higher.     

Protein oxidation in the leaves and roots of cucumber plants (Cucumis sativus L.), mutant MSC16 and wild type

Reactive oxygen species (ROS) may cause irreversible carbonylation of proteins, resulting in structural and/or functional modifications. Carbonylated proteins were analyzed and compared in tissue extracts or purified mitochondria isolated from the leaves and roots of wild-type (WT) or MSC16 mutant cucumber plants. For analysis of the oxidized protein formation and degradation, several techniques were applied: Western blotting, quantitative, spectrophotometric assay of carbonyl concentration and protease activity measurements. Oxidized proteins were tagged with 2,4-dinitrophenylhydrazine (DNPH) and detected with anti-DNP antibodies. Western blots of 1D gels indicated that, in the leaves of both WT and MSC16 plants, certain oxidized proteins have chloroplastic origin. In MSC16 plants, protein oxidation is probably higher in chloroplasts than in mitochondria. Carbonyl concentration is similar in MSC16 and WT leaf extracts, but this may be the result of twice as high protease activity observed in MSC16 leaf extracts and indicates that chloroplastic proteases may effectively remove the oxidized proteins from chloroplasts. In mitochondria of both WT and MSC16 leaves, the levels of oxidized proteins and protease activity are similar. In MSC16 root extracts, the carbonyl concentration is lower and protease activity is similar as compared to WT plants. Nevertheless, in MSC16 root mitochondria, the 30% lower carbonyl concentration, lower band abundance for oxidized proteins and over 50% higher protease activity indicate that mitochondrial proteases are involved in degradation of the oxidatively damaged proteins. In matrix and membrane subfractions, the levels of oxidized proteins are similar in leaf mitochondria or lower in root mitochondria from MSC16 as compared to WT plants. The results show that the oxidized protein degradation network in MSC16 cucumber mutants is well developed, thus becoming a survival factor for plants with mitochondrial dysfunctions.     

Determination of blood folate using acid extraction and internally standardized gas chromatography-mass spectrometry detection

Whole blood folate level is a superior indicator of folate nutritional status than serum/plasma level. Problems with and lack of confidence in results of current whole blood folate assays have limited its popularity for assessing folate nutritional status. Here, an acid extraction GCMS detection method that measures total folate whole blood is presented. Folates are released from the matrix of whole blood and cleaved to para-aminobenzoic acid (pABA) by acid hydrolysis in the presence of [(13)C(6)]pABA as internal standard (IS). The hydrolysate is passed over a C18 resin to remove heme. The pABA isotopomers are ethyl esterified, isolated on C18 resin, and trifluoroacetylated. Following normal-phase HPLC separation, the isotopomers are silylated to their tBDMS derivatives. The abundance of these derivatives are measured at m/z 324 for [(13)C(6)]pABA as IS and m/z 318 for pABA from whole blood folate. Our method uses readily available chemicals and our results agree well with those using Lactobacillus casei, the current gold standard reference assay. The presence of folate analogs (methotrexate) or antibacterials (sulfonamines) does not affect our method. This feature makes it useful in monitoring folate status of patients undergoing chemotherapy. Before using our method, pABA supplements must be discontinued for a few days.     

Tobacco-mosaic-virus-induced increase in abscisic-acid concentration in tobacco leaves:

The concentrations of free and bound abscisic acid (ABA and the presumed ABA glucose ester) increased three- to fourfold in leaves of White Burley tobacco (Nicotiana tabacum L.) systemically infected with tobacco mosaic virus. Infected leaves developed a distinct mosaic of light-green and dark-green areas. The largest increases in both free and bound ABA occurred in dark-green areas. In contrast, virus accumulated to a much higher concentration in light-green tissue. Free ABA in healthy leaves was contained predominantly within the chloroplasts while the majority of bound ABA was present in non-chloroplastic fractions. Chloroplasts from light-green or dark-green tissues were able to increase stromal pH on illumination by an amount similar to chloroplasts from healthy leaf. It is unlikely therefore that any virus-induced diminution of pH gradient is responsible for increased ABA accumulation. Tobacco mosaic virus infection had little effect on free ABA concentration in chloroplasts; the virus-induced increase in free ABA occurred predominantly out-side the chloroplast. The proportional distribution of bound ABA in the cell was not changed by infection. Treatment of healthy plants with ABA or water stress increased chlorophyll concentration by an amount similar to that induced by infection in dark-green areas of leaf. A role for increased ABA concentration in the development of mosaic symptoms is suggested.Abbreviations ABA abscisic acid - TMV tobacco mosaic virus     

NaCl胁迫对宁夏枸杞叶和幼根显微及超微结构的影响

以宁夏枸杞为材料,采用超薄切片技术制备样品,应用光学显微镜和透射电镜分析了不同浓度NaCl胁迫条件下宁夏枸杞叶和幼根显微及超微结构的变化。结果表明：随着NaCl胁迫的加重,(1)叶片上表皮细胞增厚,栅栏组织细胞出现缩短现象,排列疏松且紊乱;幼根的初生结构无明显变化。(2)叶片栅栏组织中叶绿体不再紧靠在细胞膜上,叶绿体双层膜破坏,基粒片层松散排列,杂乱无章,出现膨胀和空泡现象,淀粉粒和嗜锇颗粒增多,叶肉细胞中线粒体发生轻微变化;幼根中皮层薄壁细胞线粒体形状发生改变,结构破坏,内膜和外膜模糊甚至破裂,大多数嵴模糊,出现空泡现象;细胞核解体,基质外溢。研究表明, 不同浓度的NaCl胁迫对宁夏枸杞叶片和幼根细胞的显微及超微结构影响不同,NaCl浓度大于200 mmol/L时,宁夏枸杞叶片和幼根细胞的显微及超微结构发生了明显变化,且叶肉细胞中线粒体的变化没有叶绿体的变化显著,推测叶肉细胞中线粒体的耐盐性比叶绿体强。     

Uridinediphosphate-glucose: Isovitexin 7-O-glucosyltransferase from barley protoplasts: Subcellular localization

Protoplasts isolated from 6-d-old primary leaves of barley (Hordeum vulgare L.) contain an enzyme which transfers the glucosyl moiety of uridine-diphosphateglucose to isovitexin, resulting in the formation of saponarin, the major flavonoid of barley. Purified chloroplasts isolated from protoplasts contained less than 2% of the total glucosyltransferase activity. These chloroplasts were 97% intact, based on ribulose-bisphosphate-carboxylase activity. Similarly low levels of glucosyltransferase activity were found in mitochondria and microbody or microsomal preparations from protoplasts. The soluble fraction (cytosol) contained at least 93% of the isovitexin 7-O-glucosyltransferase activity.     

Subcellular Location of O-Acetylserine Sulfhydrylase Isoenzymes in Cell Cultures and Plant Tissues of Datura innoxia Mill

O-Acetylserine sulfhydrylase (OASS; EC 4.2.99.8) catalyzes the formation of L-cysteine from O-acetylserine and inorganic sulfide. Three OASS isoenzymes that differ in molecular mass and subunit structure are present in shoot and root tissues and in cadmium-resistant and cadmium-susceptible cell cultures of Datura innoxia Mill. Different OASS forms predominate in leaves, roots, and suspension-cell cultures. To determine the subcellular location of the OASS isoenzymes, purified mitochondria, chloroplasts, and cytosolic fractions from protoplasts were obtained. The isoenzymes are compartmentalized in D. innoxia cells, with a different isoenzyme predominant in the chloroplast, cytosol, and mitochondria, suggesting that they serve different functions in the plant cell. The chloroplast form is most abundant in green leaves and leaf protoplasts. The cytosolic form is most abundant in roots and cell cultures. A mitochondrial form is abundant in cell cultures, but is a minor form in leaves or roots. Cadmium-tolerant cell cultures contain 1.8 times as much constitutive OASS activity as the wild-type cell line, and 2.9 times more than the cadmium-hypersensitive cell line. This may facilitate rapid production of glutathione and metal-binding phytochelatins when these cultures are exposed to cadmium.     

Accumulation of chloroplast-targeted lipoxygenase in passion fruit leaves in response to methyl jasmonate

Wounding caused local and systemic induction of lipoxygenase (LOX) activity in passion fruit (Passiflora edulis f. flavicarpa) leaves, while exposing intact plants to methyl jasmonate (MJ) vapor provoked a much stronger response. Western blot analysis of these leaf protein extracts using polyclonal antibodies against cucumber LOX, revealed an accumulation of a 90 kDa protein, consistent with LOX enzymatic assays. The inducible LOX was purified to apparent homogeneity, and in vitro analysis of LOXactivity using linoleic acid as substrate showed that it possesses C-13 specificity. Immunocytochemical localization studies using leaf tissue from MJ-treated plants demonstrated that the inducible LOX was compartmented in large quantities in the chloroplasts of mesophyll cells, associated with the stroma. The results suggest that the wound response in passion fruit plants may be mediated by a chloroplast 13-LOX, a key enzyme of the octadecanoid defense-signaling pathway.     

ESTERASE INHIBITION BY ORGANO-PHOSPHORUS RESIDUES, WITH SOME OBSERVATIONS ON POSSIBLE EFFECTS ON PLANT METABOLISM

Organo-phosphorus residues in tissues of sprayed plants were detected by estimating the esterase-inhibiting activity of their leaf and root extracts. This method was used to examine the anti-esterase effects of mangold plants that had been sprayed with Systox, parathion and Hanane. Extracts of leaves of these treated plants were shown to inhibit added choline esterase for some weeks after treatment. The enzymic hydrolysis of phenyl acetate by extracts of leaves and roots of mangold plants treated with these insecticides was reduced for varying periods of up to 8 weeks.
Assays of parts of bean plants that had been sprayed with demeton-S showed that anti-esterase activity was limited to those parts that had been sprayed: tissues developed subsequent to spraying showed no such effects. Leaves sprayed about z months previously inhibited added choline esterase and showed reduced activity in hydrolysing phenyl acetate. There is some evidence that substances, possibly substrates in the plant enzyme systems affected, accumulate in treated leaves.     

Enzymes of starch metabolism in leaves and berries of Vitis vinifera

Leaves of Vitis vinifera L., cv. Cabernet Sauvignon contained 2.0 mg of starch per g fresh weight, whereas young green berries and maturing grape berries contained less than 0.03 mg of starch, despite the presence of abundant substrates (reducing sugars and sucrose) in berries for starch synthesis. the activities of several enzymes likely to be involved in starch synthesis were determined in extracts of berries and leaves. Fractionation procedures resulted in final recoverable ADPglucose-starch glucosyltransferase activity which was 2–3 times the activity measured in crude extracts of leaves. Compared to leaves, berries contained low activities of ADPglucose-starch glucosyltransferase and ADPglucose pyrophosphorylase. These enzymes increased only 2- to 3-fold from young to maturing berries. ADPglucose-starch glucosyltransferase activity in the absence of added primer was found in leaf extracts but not in berry extracts. The activities of UDP-glucose pyrophosphorylase, phosphorylase and amylase were comparable in both leaves and berries and increased 6- to 7-fold during berry development. The low activities of ADPglucose-starch glucosyltransferase and ADPglucose pyrophosphorylase probably account for the paucity of starch in grape berries.     

CHLOROPLAST STRUCTURE AND FUNCTION IN CULTURED TOBACCO TISSUE

A comparative study was made of the ability of cultured pith tissue, leaves of buds induced from callus, and mature leaf tissue of Nicotiana tabacum L. ‘Maryland Mammoth’ to fix carbon, as determined by light-induced C¹⁴O₂ incorporation. Photosynthetic ability was then correlated with the fine structure of chloroplasts from these tissues. The light to dark incorporation ratio for C¹⁴O₂ was at least 3 times as great in the leaf tissue as in growing cultured tissue. The chlorophyll content of the leaf tissue was 10 times as great. The carbon fixation pattern of all the tissues, as determined by radioautographs of chromatogramed extracts, was qualitatively the same. The rate of sucrose synthesis differed greatly, since 20% of the total radioactivity of the extracts from mature leaf tissue appeared in sucrose, while only 1.0% was found in sucrose from callus extracts. The incorporation of C¹⁴O₂ into sugars was inhibited in all the tissue by DCMU (3,4-dichlorophenyl,1, 1-dimethylurea). Cultured tissue past the log phase of growth was intermediate between the younger cultured tissue and the leaf tissue in its chlorophyll content and ability to incorporate C¹⁴O₂ in the light. Proplastids from dark-grown callus possessed stroma lamellae, but prolamellar bodies were not observed. The chloroplasts from growing callus were partially differentiated in comparison with chloroplasts from mature leaf tissue, since each granum had only 4-7 lamellae. Chloroplasts from callus past the log phase of growth were indistinguishable from those in mature leaves. This study establishes that the differentiation of chloroplasts in cultured tissue is a function of the growth rate of the tissue. The growth rate and degree of differentiation of the tissue can be regulated, so a well-defined system is available for the experimental study of chloroplast differentiation.     

The nucleotide sequence of a complementary DNA encoding Flaveria bidentis carbonic anhydrase

We have isolated and characterised a cDNA clone encoding the cytosolic form of carbonic anhydrase in the leaves of Flaveria bidentis, a C₄ dicotyledonous plant. The deduced amino acid sequence is similar to the carbonic anhydrase found in the chloroplasts of C₃ dicotyledonous plants. Western blot analysis of crude leaf extracts of F. bidentis indicates that the leader sequence (equivalent to the transit peptide of the chloroplastic form of CA found in C₃ plants) is not removed following translation of mRNA.     

Hydrolysis of diethyl diferulates by a tannase from Aspergillus oryzae

Diferulic acid forms cross-links in naturally occurring plant cell wall polymers such as arabinoxylans and pectins. We have used model ethyl esterified substrates to find enzymes able to break these cross-links. A tannase from Aspergillus oryzae exhibited esterase activity on several synthetic ethyl esterified diferulates. The efficiency of this esterase activity on most diferulates is low compared to that of a cinnamoyl esterase, FAEA, from Aspergillus niger. Of the diferulate substrates assayed, tannase was most efficient at hydrolysing the first ester bond of the 5–5- type of dimer. Importantly and unlike the cinnamoyl esterase, tannase from A. oryzae is able to hydrolyse both ester bonds from the 8–5-benzofuran dimer, thus forming the corresponding free acid product. These results suggest that tannases may contribute to plant cell wall degradation by cleaving some of the cross-links existing between cell wall polymers.     

Toxic tetrapyrrole accumulation in protoporphyrinogen IX oxidase-overexpressing transgenic rice plants

We generated transgenic rice plants (Oryza sativa cv. Dongjin) over-expressing human protoporphyrinogen IX oxidase (PPO) with the aim to increase mitochondrial PPO activity and confer herbicide resistance (Lee et al., Pestic Biochem Physiol 80:65-74, 2004). The transgenic plants showed during further leaf development the formation of severe necrotic spots and growth retardation. Several experiments were performed to examine the reasons for the formation of necrotic leaf lesions. Human PPO is normally located in mitochondria. An in vitro organellar import experiment revealed translocation of human PPO into pea chloroplasts, but not into mitochondria. Using a specific antibody raised against human PPO confirmed its plastidic localisation. The heme and chlorophyll contents were lower in necrotic leaves than wild-type leaves. Interestingly, mature and necrotic leaves of 12-week-old transgenic plants contained up to 14- and 24-fold more protoporphyrin IX, respectively, than mature wild-type leaves. Enhanced levels of Mg-Protoporphyrin IX, Mg-Protoporphyrin IX monomethyl ester and protochlorophyllide were concurrently observed in transgenic plants relative to wild type. Accumulated porphyrins and Mg-porphyrins likely act as photosensitizers and cause high formation of the reactive oxygen species. These high levels of tetrapyrrole intermediates correlated with increased rates of 5-aminolevulinic acid synthesis in transgenic plants. Tetrapyrrole-induced photooxidation was confirmed by increased lipid peroxidation and subsequent cell death. The transgenic phenotype is the consequence of a highly modified tetrapyrrole metabolism due to additional expression of human PPO. A possible regulatory role of PPO in graminaceous seedlings is discussed.