Flavonoid Responses in Wheat Grown at Elevated CO2: Green Versus Senescent Leaves

We compared flavonoids in green, mature, and senescing flag leaves of wheat grown under ambient (AC - 370 mol mol^-1) and elevated (EC - 550 mol mol^-1) concentrations of CO₂ in a FACE (Free Air CO₂ Enrichment) system. The concentrations of flag leaf flavonoids (e.g., isoorientin and tricin) decreased to one third in mature leaves, and the majoritary isoorientin almost disappeared in senescing leaves. Flavonoid concentrations increased in green well-developed flag leaves under EC (46 % isoorientin and 55 % tricin), whereas the differences disappeared in mature and senescing flag leaves. Predictions of changes in litter phenolic concentrations and their effects on decomposition rates under EC based on changes in green leaves need to be revised.     

Seasonal changes in oleosomic lipids and fatty acids of perennial root nodules of beach pea

Seasonal changes in the fatty acid composition of phospholipids (PL), monoglycerides (MG), diglycerides (DG), free fatty acids (FA) and triglycerides (TG) separated from oleosomes (lipid bodies) of perennial root nodules of beach pea (Lathyrus maritimus) were analysed. Thin layer chromatography (TLC) revealed that PL and MG are the major lipids in nodule oleosomes. The fatty acid profile and overall double bond index (DBI) varied among lipid classes depending upon the season. High DBI in PL and MG found during late winter and early spring indicated that they may play a major role in winter survival and regeneration of perennial nodules. The DBI of DG was high at the end of the fall season and the DBI of FA and TG was high in summer months. The dominant fatty acids are C16:0 followed by C18:0 and C18:1. The levels of many unsaturated fatty acids such as C18:1, C18:2 and C18:3 increased while saturated fatty acid C18:0 decreased during winter. These unsaturated fatty acids possibly play an important role in the protection of nodule cells from cold stress. Nodules seem to retain some fatty acids and selectively utilize specific fatty acids to survive the winter and regenerate in spring.     

Peroxisomal enzyme activities in attached senescing leaves

Recently it has been demonstrated that detached leaves show glyoxysomal enzyme activities when incubated in darkness for several days. In this report glyoxylate-cycle enzymes have been detected in leaves of rice (Oryza sativa L.) and wheat (Triticum durum L.) from either naturally senescing or dark-treated plants. Isolated peroxisomes of rice and wheat show isocitrate lyase (EC 4.1.3.1), malate synthase (EC 4.1.3.2) and -oxidation activities. Leaf peroxisomes from dark-induced senescing leaves show glyoxylic-acid-cycle enzyme activities two to four times higher than naturally senescing leaves. The glyoxysomal activities detected in leaf peroxisomes during natural foliar senescence may represent a reverse transition of the peroxisomes into glyoxysomes.This work was supported by CNR Italy, special grant RAISA, subproject 2, paper no. 26.     

Medium-chain fatty acid biosynthesis and utilization in Brassica napus plants expressing lauroyl-acyl carrier protein thioesterase

We have examined production of mediumchain fatty acids by Brassica napus L. plants transformed with a California bay (Umbellularia californica) medium-chain acyl-acyl carrier protein (ACP) thioesterase (UcFatB1) cDNA under the control of the constitutive cauliflower mosaic virus 35S promoter. These plants were found to accumulate medium-chain fatty acids in seeds but not in leaves or roots. Assay of thioesterase activity in extracts of leaves indicated that lauroyl-ACP thioesterase activity is comparable to oleoyl-ACP thioesterase (EC 3.1.2.14) activity in transformant leaves. Furthermore, leaf lauroyl-ACP thioesterase activity was in excess of that which produced a significant increase in the amount of laurate (12:0) in seed. Studies in which isolated chloroplasts were ¹⁴C-labelled were used to evaluate whether medium-chain fatty acids were produced in transformed leaves. Up to 34% of the fatty acids synthesized in vitro by isolated chloroplasts were 12:0. These results demonstrate that the normally seed-localized lauroyl-ACP thioesterase can be expressed in active form in leaves, imported into chloroplasts and can access acyl-ACP intermediates of leaf de-novo fatty acid synthesis. The most likely explanation for the lack of accumulation of 12:0 in transformed leaves is its rapid degradation by -oxidation. In support of this hypothesis, isocitrate lyase (EC 4.1.3.1) activity was found to be significantly increased in plants transformed with 35S-UcFatB1.Abbreviations ACP acyl carrier protein - CaMV cauliflower mosaic virus - control Brassica napus cultivar 212/86 - event 8 pCGN3831-212/86-8 - event 11 pCGN3831-212/86-11 - FAS fatty acid synthase - IL isocitrate lyase - KAS -keto-acyl ACP synthase - MS malate synthase - OTE oleoyl-ACP thioesterase - TAG triacylglycerol - UcFatB1 California bay medium-chain acyl-ACP thioesterase We are indebted to Calgene's Brossica-transformation, growth-chamber, greenhouse, and lipid-analysis personnel. Maelor Davies conducted the initial tranformant analysis. We thank Laura Olsen for IL and MS Western blot analysis and advice on IL and MS activity assays. This work was supported in part by a grant from the U.S. Department of Energy (No. DE-FG02-87ER12729). Acknowledgement is made to the Michigan Agricultural Experiment Station for its support of this research.     

Seed oil fatty acid patterns of theAconitum-Delphinium-Helleborus complex (Ranunculaceae)

Many members ofRanunculaceae contain unusual fatty acids in their seed oils. This leads to rather typical genus-specific fatty acid patterns or fingerprints in these seed oils. The members of theDelphinioideae and/orHelleboroideae, however, do not contain highly unusual fatty acids. Nevertheless, their seed oil fatty acid fingerprints are also fairly typical and genus-specific, and the patterns found are rather consistent throughout several species of one genus. It was found that species ofAconitum do not contain fatty acids with 20 carbon atoms.Delphinium, Consolida, Helleborus, Nigella and others do contain C₂₀ fatty acids. In allHelleborus species, for example, there was a consistent C₂₀ fatty acid pattern of 20:020:120:2>20:3. Species ofNigella andGaridella contain high levels,Helleborus low levels, of 20:2n-6 in their seed oils.Delphinium andAconitum both contain low levels of 18:3n-3, whereasHelleborus spp. consistently show high levels of this fatty acid. The genus-specific fatty acid patterns found are discussed, and a correlation with the subfamily and tribe affiliation of the genera investigated here is attempted.     

Leaf rolling effects on lipid and fatty acid composition in Ctenanthe setosa (Marantaceae) subjected to water-deficit stress

Vegetatively propagated Ctenanthe setosa (Rosc.) Eichler (Marantaceae) plants were grown in plastic pots under laboratory irrigation and water deficit conditions. One set of plants was submitted to water irrigation regularly and another set of plants was submitted to water deficit conditions. After a 28 d water deficit stress, the leaves started to roll. Approximately after 33–35 d, the leaves were tightly rolled. Water stress significantly increased the dry weight of rolled leaves. After the 35 d period of water deficit the open (non-stressed) and rolled (stressed, water deficit) leaves were harvested for lipid content and class compositional analysis. The fatty acids consistently identified in phospholipids and glycolipids as well as in total leaf lipid were 16:0, 18:0, 18:1, 18:2 and 18:3. The 16:0, 18:3 and 18:1 acids in control plant and 18:2, 16:0 and 18:3 acids in rolled leaves were determined as the major fatty acids. While the percentage composition of 16:0, 18:1 and 18:3 acids decreased in rolled leaves, the level of 18:2 acid increased. However, the percentage composition of unsaturation in phospholipid (71%) and glycolipid (80.4%) fractions in rolled leaves were found higher than in control leaves. The results show that the degree of unsaturation in phospholipid, glycolipid and total lipid was significantly altered during leaf rolling. The increase in unsaturation degree may regulate membrane permeability and thus adapt the leaves to water stress in the drought environment.     

Fatty acids,starch and biomass of Scots pine needles and roots in open-air ozone exposure

Scots pine (Pinus sylvestris L.) seedlings were fumigated with 1.2–1.5 x ambient ozone (cumulative exposure) over 2 seasons in an open-air experiment. Starch and fatty acid concentrations were analyzed in needle and root tissue in the summer, autumn and early winter. Seedling growth was determined by measuring the height of the stem and the total shoot and root biomass. Significant decreases in growth were found in exposed seedlings, even though visible symptoms were lacking. Almost significant reductions in needle and root starch concentrations were found. In the ozone treated foliage, significant increases in myristic acid (140) were detected, but the major fatty acids remained unchanged. Fatty acid ratios showed that the degree of unsaturation decreased in treated needles in the summer. In the roots of ozone treated seedlings, changes in fatty acids were different from those in the foliage. Decreases of the main root fatty acids (160, 180, 181, 18:2, 183) were detected in the summer. These results show that Scots pine is susceptible to enhanced levels of ozone. If the tropospheric ozone levels continue to increase it may have deleterious effects on Scots pine forests in Finland.     

Triacylglycerols in the seeds of northern Scots pine,Pinus sylvestris L., and Norway spruce,Picea abies (L.) Karst.

The geographical variation in the composition of triacylglycerols in seeds of Scots pine (Pinus sylvestris L.) and Norway spruce [Picea abies (L.) Karst.], grown in Finland, was analyzed. The total lipid content of pine seeds was slightly higher in the northernmost provenance (68 °50N), whereas the lipid content of spruce seeds was not affected systematically by the geographical origin of the seeds. The species studied differed in the proportions of fatty acids in their triacylglycerols, though the three most abundant components were the same, i.e. oleic acid (181 n9), linoleic acid (182n-6) and 5,9,12-octadecatrienoic acid (183 5c9c12c). These fatty acids corresponded to more than 80 mol% of the total fatty acids. According to mass spectrometric analyses, the triacylglycerols of both spruce and pine seeds consisted of the same molecular species with 52–56 acyl carbons, but in different proportions. Molecules with 54 acyl carbons represented approximately 75% of the pine and 85% of the spruce triacylglycerols, with the most abundant molecular species being 545, 546 and 547. Some minor differences in the fatty acid composition of triacylglycerols of pine seeds from different seed collecting areas were found: the proportion of linoleic acid slightly increased whereas that of 5,11,14-eicosatrienoic acid decreased towards the northern origins. Similar differences were not found in the proportions of fatty acids in spruce seed lots. Furthermore, the proportions of triacylglycerols in both pine and spruce seeds from northern and southern collecting areas were not significantly different. The higher content of total lipids in spruce seeds compared with pine seeds may be due to the structure of the seed coat, and the lipophilic layers inside it, acting as a barrier to imbibition.     

Relationship between contents of leucoanthocyanidin and dhurrin in sorghum leaves

Summary Flag leaves of Colman forage sorghum (Sorghum bicolor) contain at least 25 times as much leucoanthocyanidin (LAC) and approximately half as much of the cyanogenic glucoside, dhurrin, as do flag leaves of White Collier forage sorghum. Assays of flag leaves from 119 F₂ plants and 11 F₅ lines from crosses between these two cultivars revealed a statistically significant negative association between levels of LAC and dhurrin. Both LAC and dhurrin are aromatic compounds, and the negative association between the two may be the result of competition for intermediates or products of the aromatic biosynthetic pathway. This rationale appears to be quite different from that for the negative association reported for levels of tannin and cyanide in Lotus corniculatus. Although the negative relationship between LAC and dhurrin in sorghum was statistically significant, the association was not consistent enough to suggest that either trait could be used reliably in selecting or breeding to modify the other trait.Contribution from USDA-ARS and the University of Nebraska Agric. Res. Div. Published as Paper No. 8003, Journal Series, Agric. Res. Div. The work reported was done under Agric. Res. Div. Project 12–114     

Lipochemistry of the progamic stage of a self-incompatible species: Neutral lipids and fatty acids of the secretory stigma during its glandular activity,and of the solid style,the ovary and the anther in Forsythia intermedia Zab. (Heterostylic species)

Chromatographic (thin-layer, gas column, column chromatography) analyses of neutral lipids and fatty acids of reproductive tissues of Forsythia intermedia Zab., a self-incompatible species, were performed with two objectives in mind: 1. To determine whether there is a qualitative evolution of the different classes of lipids and fatty acids that could be correlated with the three functional stages observed during previous histochemical and ultrastructural studies. The stigmatic exudate and intracellular accumulations consist mainly of neutral lipids. 2. To compare the lipid composition of the stigma (both thrum and pin forms) with that of the style, the ovary, and the anther, and to investigate the possible existence of a stigma-specific lipid compound. Stigmatic neutral lipids are found mostly in a glyceridic mixture probably containing hydrocarbons and terpenes. The fatty acids identified are between C:7 and C: 12, with the maximum unsaturated form being a C: 18. During the secretory process there is no great qualitative diference between the neutral lipids and fatty acids found in the stigmas of thrum and pin forms. Sterols are present in styles, ovaries, and anthers, but not in stigmas. They represent the only difference in the lipid composition of these various floral structures.     

Free tryptophan and indole-3-acetic acid levels in the leaves and vascular pathways of Ricinus communis L.

Levels of free tryptophan in the leaves, phloem and xylem saps of Ricinus communis L. were determined by colorimetric assay. Values of 0.38 g ml^-1 in root pressure sap and 96.0 g ml^-1 in phloem sap were recorded. Tryptophan levels were highest in mature and senescing leaves. Levels of indoleacetic acid (IAA) in the phloem sap and leaves were determined by gas chromatography—mass spectrometry using a deuterated internal standard. A mean value of 13.0 ng ml^-1 was recorded in phloem sap. The distribution in the leaves showed an inverse relationship to that of tryptophan, being highest in young leaves.Abbreviations IAA indoleacetic acid - GC-MS Gas chromatography-mass spectrometry - PFP-derivative pentafluoropropionyl-derivative - TLC thin layer chromatography     

A senescence-associated gene of Arabidopsis thaliana is distinctively regulated during natural and artificially induced leaf senescence

We have characterized the structure and expression of a senescence-associated gene (sen1) of Arabidopsis thaliana. The protein-coding region of the gene consists of 5 exons encoding 182 amino acids. The encoded peptide shows noticeable similarity to the bacterial sulfide dehydrogenase and 81% identity to the peptide encoded by the radish din1 gene. The 5-upstream region contains sequence motifs resembling the heat-shock- and ABA-responsive elements and the TCA motif conserved among stress-inducible genes. Examination of the expression patterns of the sen1 gene under various senescing conditions along with measurements of photochemical efficiency and of chlorophyll content revealed that the sen1 gene expression is associated with Arabidopsis leaf senescence. During the normal growth phase, the gene is strongly induced in leaves at 25 days after germination when inflorescence stems are 2–3 cm high, and then the mRNA level is maintained at a comparable level in naturally senescing leaves. In addition, dark-induced senescence of detached leaves or of leaves in planta resulted in a high-level induction of the gene. Expression of the sen1 gene was also strongly induced in leaves subjected to senescence by 0.1 mM abscisic acid or 1 mM ethephon treatment. The induced expression of the gene by dark treatment was not significantly repressed by treatment with 0.1 mM cytokinin or 50 mM CaCl₂ which delayed loss of chlorophyll but not that of photochemical efficiency.     

Effect of iron chlorosis on mineral nutrition and lipid composition of thylakoid biomembrane in Prunus persica (L.) Bastch.

The effect of iron chlorosis on mineral, thylakoid lipids and fatty acids composition of field grown peach tree leaves was studied. Significant differences were found in iron extracted by using , -dipyridyl (active iron), total iron, P, K, Cu and the P/Fe and Fe/Mn ratios. The levels of total chlorophyll, total glycolipids and phospholipids were reduced under iron chlorosis. A slight iron deficiency does not modify the fatty acid composition of thylakoid membranes, while a strong deficiency changes the proportion of some fatty acids.Abbreviations Chl chlorophyll - DGDG digalactosyldiglycerol - MGDG mono-galactosyldiglycerol - PC phosphatidycholine - PE phophatidylethanolamine - PG phophatidylglycerol - TLC thin layer chromatography - 16:0 palmitic acid - 16:1 palmitoleic acid - 16:lt trans-hexadecenoic - 18:0 steric acid - 18:1 oleic acid - 18:2 linoleic acid - 18:3 linolenic acid     

Identification of conserved domains in the Δ12 desaturases of cyanobacteria

Cyanobacterial genes for enzymes that desaturate fatty acids at the 12 position, designated desA, were isolated from Synechocystis PCC6714, Synechococcus PCC7002 and Anabaena variabilis by crosshybridization with a DNA probe derived from the desA gene of Synechocystis PCC6803. The genes of Synechocystis PCC6714, Synechococcus PCC7002 and A. variabilis encode proteins of 349, 347 and 350 amino acid residues, respectively. The transformation of Synechococcus PCC7942 with the desA genes from Synechocystis PCC6714, Synechococcus PCC7002 and A. variabilis was associated with the ability to introduce a second double bond at the 12 position of fatty acids. The amino acid sequence of the products of the desA genes revealed the presence of four conserved domains. Since one of the conserved domains was also found in the amino acid sequences of 3 desaturases of Brassica napus and mung bean, this domain may play an essential role in the introduction of a double bond into fatty acids bound to membrane lipids.Abbreviations X:Y(Z) fatty acid containing X carbon atoms with Y double bonds in the cis configuration at position Z counted from the carboxyl terminus     

Membranous appendices of spherosomes (oleosomes)

Spherosomes (oleosomes) of cotyledons of rape (Brassica napus L.), sunflower (Helianthus annuus L.), and watermelon (Citrullus vulgaris, Schrad.) seedlings are delimited by a half unit membrane that appears to be continuous with each of the osmiophilic layers of a tripartite unit membrane forming a handlelike appendix of the spherosomes. Prior to any noticeable utilization of the spherosomal storage fat, ribosomes were found to be attached to these handles. At later stages appendices of the spherosomes are smooth, showing a diameter of about 22 nm that greatly exceeds the thickness of any other unit membrane profiles identical in structure and diameter osomes appears to be continuous with the thick lipid layer of the handles. In intermediate stages of fat depletion the spherosomal bodies become invaginated with cytoplasmic material. Finally vesicles with cytoplasmic contents surrounded by a membrane with a typically thick lipid layer are left in the cells. Membrane profiles indentical in structure and diameter to the spherosomal appendices were also present in electron micrographs of the lipolytic membrane fraction recovered from sucrose density gradients after centrifugation of a microsomal cell fraction. The ultrastructural observations are taken for evidence that the spherosomal appendices represent the lipase-carrying membranes isolated previously (Theimer and Rosnitschek, 1978). A novel hypothesis for development and utilization of fat-storing spherosomes is also proposed.     

Occurrence of Curtobacterium sp. possessing ω-cyclohexyl fatty acids in soil with zinc added

A bacterial strain, Curtobacterium sp., isolated from a soil with zinc added possessed -cyclohexyl fatty acids. -Cyclohexyl undecanoic acid made up 47% of the total fatty acids; it was the most abundant fatty acid in the strain grown in tryptone medium. 12-Methyl tetradecanoic acid (23%) and 14-methyl hexadecanoic acid (22%) were also major fatty acids. The proportion of -cyclohexyl undecanoic acid increased as the pH of the medium decreased and as the culture temperature increased.The bacteria grew almost normally in zinc-enriched medium, and -cyclohexyl undecanoic acid increased with zinc concentration. Zinc added to the medium was not abundant in the cell fraction, and the ratio of increase of zinc in the cells was not so high as in the culture medium. These results suggested that -cyclohexyl fatty acids are related to the zinc tolerance of the isolated strain, and that this tolerance depends on low permeability of the membrane to zinc.     

Genetic control of plastid carotenoids and transformation in the skin of Cucurbita pepo L. fruit

Summary The influence of allelic state of gene B on skin pigmentation in two cultivars of Cucurbita pepo L. has been studied. Total carotenoids were lower at early stages of fruit development in cultivar (cv.) Early Prolific (EP) BB YY fruit skin, than in EP B ⁺ B ⁺ YY fruit skin, but no differences were observed in total skin carotenoids twenty days after anthesis. Total carotenoids were lower in cv. Fordhook Zucchini (FZ) BB yy fruit skin, than in FZ B ⁺ B ⁺ yy fruit skin at all developmental stages from anthesis to maturity. Both green and yellow tissues contained typical foliar carotenoids. The carotenoids from yellow fruit skin of both EP genotypes and of FZ BB were characterized by a low carotene: xanthophyll ratio, with a high proportion of the xanthophylls esterified to fatty acids. The xanthophylls of the yellow tissues were esterified with 120, 140, 160 fatty acids. The carotenoids from the green fruit skin of FZ B ⁺ B ⁺ had a higher percentage of carotenes (primarily -carotene) and a lower percentage of esterified xanthophylls. Spectral shapes of carotenoid fractions from all yellow tissues were similar and distinguishable from those of green FZ B ⁺ B ⁺ tissue. The results of these studies are discussed in terms of the genetic control of plastid transformation in Cucurbita pepo L.New Jersey Agricultural Experiment Station No. D99201 (NE-9) 32-83, supported by state funds and funds from the Rutgers University Research Council     

Biochemical composition of three algal species proposed as food for captive freshwater mussels

To identify potential diets for rearing captive freshwater mussels, the protein, carbohydrate (CHO), and lipid contents of two green algae, Neochloris oleoabundans, Bracteacoccus grandis, and one diatom, Phaeodactylum tricornutum, were compared at different growth stages. The fatty acid and sterol composition were also identified. Protein was greatest (55–70%) for all species at late log growth stage (LL), and declined in late stationary (LS) growth. CHO was greatest at LS stage for all species (33.9–56.4% dry wt). No significant change in lipid levels occurred with growth stage, but tended to increase in N. oleoabundans. Mean lipid content differed significantly in the order: N. oleoabundans > P. tricornutum > B. grandis. Total fatty acids (TFA) were higher at LS stage compared to other stages in the two green algae, and stationary stage in the diatom. Mean unsaturated fatty acids (UFA) as %TFA was significantly higher in N. oleoabundans than the other species. The green algae contained high percentages of C-18 polyunsaturated fatty acids (PUFAs), while the diatom was abundant in C-16 saturated and mono-unsaturated fatty acids and C-20 PUFA fatty acids. Growth stage had no effect on sterol concentration of any species. B. grandis showed significantly higher sterol levels than the other species except P. tricornutum at S stage. B. grandis was characterized by predominantly ⁵, C-29 sterols, while N. oleoabundans synthesized ^5,7, ^5,7,22 , and ⁷, C-28 sterols. P. tricornutum produced primarily a ^5,22, C-28 sterol, and a small amount of a ^7,22, C-28 sterol.     

Oleosomes (lipid bodies) in nitrogen-fixing peanut nodules

Abstract. Nitrogen-fixing peanut root nodules have oleosomes (lipid bodies) in the infected cells. The oleosomes have been characterized and compared with their counterparts in the seed tissue. Eighty per cent of the nodule oleosomes were found to be of smaller size (0.16–1.0 μm diameter [dia.]) while in the seeds the larger size (2.0–5.5 μm dia.) dominated. The larger oleosomes were exclusively found in the uninfected three layers of cortical cells adjacent to the infected zone. Morphometric analyses have revealed significantly higher numbers of oleosomes covering about 8% of the infected cell area in immature (white) nodules, whereas the mature/old (pink) nodules had lesser numbers occupying about 4–5% of the cell area. The decrease in the amount of oleosomes possibly reflects their utilization in mature/old nodules which effectively fix nitrogen. The oleosomes were distinctly stained by p-phenylenediamine (pPD) at both light and electron microscopic levels. An electron-dense rim was observed around the nodule oleosomes; where lipolytic activity was also demonstrated using cytochemical methods. The rim was absent in the seed oleosomes. The defatted oleosomes were found to be surrounded by a 'half unit membrane' and a non-extractable rim of possible pro-teinaceous substance. Gas chromatographic analyses of the lipid from the isolated oleosomes indicated the presence of higher amounts of saturated fatty acids in the nodule oleosomes than the ones in the seed. The study indicates that the nodule oleosomes differ from seed oleosomes, with respect to the presence of (1) an electron-dense rim showing (2) lipolytic activity and (3) higher amounts of saturated fatty acids. Nodule oleosomes seem to be transient storage organelles to be metabolized, while in the seed they are meant for long-term storage.