Lipid metabolism in green leaves of developing monocotyledons

Lipid synthesis was studied in successive leaf sections from the base to the tip of developing barley (Hordeum vulgare L.), maize (Zea mays L.), rye grass (Lolium perenne L.) and wheat (Triticum aestivum L.) leaves. The endogenous levels of acyl lipids and their constituent fatty acids from the same leaf sections were also analysed. The principle chloroplast acyl lipids showed a relative increase in amount with the age of the leaf section. Their content of -linolenic acid also increased whereas there was little change in the amount of this acid in phosphatidylcholine and phosphatidylethanolamine, which are primarily non-chloroplastic. The content of trans-3-hexadecenoic acid in phosphatidylglycerol increased approximately 20-fold between the youngest (basal) and oldest (distal) leaf sections.The incorporation of [¹⁴C]acetate was always high into monogalactosyldiacylglycerol, phosphatidylcholine and the neutral lipid (mainly pigments) fractions. With increasing age, the neutral lipids were less well labelled. In three of the plant species but not in barley, phosphatidylglycerol was heavily labelled. Monogalactosyldiacylglycerol usually contained the highest amount of radioactivity in the middle leaf sections. Apart from these generalisations, each plant type had its own specific pattern of radiolabelling.     

Sequential changes in the lipids of developing proplastids isolated from green maize leaves

Changes in lipid composition were followed as a proplastid develops into a chloroplast. Methods were devised for the isolation of developing proplastids from sections of five different ages from the same 7-day-old maize (Zea mays var. Kelvedon Glory) leaf. Electron micrographs illustrate the homogeneity of the five types of plastid suspension, minimal contamination with other cytoplasmic membranes, and the presence of morphologically intact plastids in the proportions 85% (youngest), 85%, 80%, 70% and 60% (oldest), respectively. Both bundle sheath and mesophyll plastids are well preserved in isolation. Plastid numbers were determined from calibration curves of the chlorophyll content of each type of suspension, and lipid values then expressed as nmoles/10⁶ plastids. Monogalactosyl diglyceride (MGDG), digalactosyl diglyceride (DGDG), sulfoquinovosyl diglyceride, and phosphatidyl glycerol (PG) all increase during plastid development but the rate of increase is different for each lipid. The largest changes are in MGDG (6-fold) and DGDG (4-fold). Phosphatidyl choline shows a continuous decline during plastid development. Phosphatidyl inositol and phosphatidyl ethanolamine were found in all the suspensions in low concentrations (0.4-4.0% of total lipid): calculations showed their presence could not be accounted for by bacterial or mitochondrial contamination. The increase in PG parallels the chlorophyll changes during development and at maturity 1 molecule of PG is present per 3 molecules of chlorophyll. The results are discussed in the context of the molecular structure of the photosynthetic thylakoid membranes.     

Lipid biosynthesis in developing perilla seeds

In developing seeds of Perilla frutescens var. crispa, the triacylglycerol fraction was found to accumulate between 15 and 19 days after flowering. Of this, 65% of the total fatty acids was alpha-linolenic acid in the mature seeds, with the latter being esterified in comparable amounts at all positions (sn-1, 2 and 3) of the glycerol residue. It was also demonstrated that, 1-acylglycerol-3-phosphate acyltransferase, which catalyzes esterification at the sn-2 position of the glycerol backbone, showed low activities for alpha-linolenoyl-CoA as substrate. These findings suggest that the diacylglycerol precursor for triacylglycerol synthesis is not directly derived from phosphatidic acid through the glycerol phosphate pathway.     

Plastid differentiation, acyl lipid, and Fatty Acid changes in developing green maize leaves

Plastid differentiation, acyl lipid, and fatty acid composition have been followed in successive 2-cm sections from the base (youngest tissue) to the tip (oldest tissue) of green Zea mays (maize) leaves grown under a normal diurnal light regime. Although the youngest cells (0-4 cm from the leaf base) had only proplastids with one or two grana, they contained chlorophylls a and b, monogalactosyldiglyceride, digalactosyldiglyceride, sulfolipid, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol. In the more mature sections, the plastids increased in size 5-fold, and differentiation into mesophyll and bundle-shealth chloroplasts had occurred. Concomitantly, the levels of all the lipids increased with the exception of phosphatidylcholine and phosphatidylethanolamine which decreased. With increasing cell maturity, the percentage of linolenic acid increased in all the individual acyl lipids, but palmitic acid remained constant in phosphatidylcholine, phosphatidylethanolamine, and sulfolipid. The Δ^3t-hexadecenoic acid was only detectable in the phosphatidylglycerol of the most mature maize tissue.     

Lipid biosynthesis by chloroplasts from olive tree leaves

In vitro incubation of isolated chloroplasts from young olive tree leaves ( Olea europaea L. cv. Marteño) in acetate-1-¹⁴C showed a high labelling of saturated fatty acids (palmitic + stearic) and, above all, of the monounsatured ones (oleic); the low biosynthetic rate of α-linolenic acid being noteworthy. These fatty acids are mainly found as free ones, or incorporated in mono-and diglyceride molecules. Phosphoand galactolipids, the most abundant acyl-lipid components of chloroplast lamellae, showed low incorporation rates. The fatty acid synthesis by isolated chloroplasts depends on exogenous CoA, ATP, NADPH and, especially, on added ACP (acyl carrier protein) preparation from Escherichia coli , whereas it was strongly inhibited by Triton X-100.
In vivo experiments with acetate-1-¹⁴C infiltration into young excised leaves showed a high labelling of chloroplast phospholipids, but a low ¹⁴C incorporation into galactolipids, a remarkable feature because these latter are main components of chloroplast lamellae. The high biosynthetic rate of α-linolenic acid is noteworthy and appears mainly linked to monogalactosyldiglycerides. Also the low incorporation of saturated fatty acids to neutral lipids is remarkable. The low in vitro synthesis of α-linolenic acid in comparison with that of the in vivo conditions, suggests the existence of a cooperation between chloroplasts and other parts of the cell to carry out the synthesis of this compound.     

The appearance of photosynthetic proteins in developing maize leaves

Soybean (Glycine max (L.) Merr.) protoplasts have been surface-labelled with cationized ferritin, and the fate of the label has been followed ultrastructurally. Endocytosis of the label occurs via the coated-membrane system. The pathway followed by the label, once it has been taken into the interior of the protoplast, appears to be similar to that found during receptor-mediated endocytosis in animal cells. Cationized ferritin is first seen in coated vesicles but rapidly appears in smooth vesicles. Labelled, partially coated vesicles are occasionally observed, indicating that the smooth vesicles may have arisen by the uncoating of coated vesicles. Structures which eventually become labelled with cationized ferritin include multivesicular bodies, dictyosomes, large smooth vesicles, and a system of partially coated reticula.Abbreviation CF cationized ferritin     

Ultrastructural detection of sucrose synthase distribution in developing maize leaves

Summary A developing maize leaf grows by the activity of a basal meristematic region and an adjacent elongating zone, resulting in a morphological and functional gradient along the leaf. We have used this system to detect the spatial and temporal expression of an enzyme, sucrose synthase, which plays a pivotal role in the sucrose import-export transition which occurs along a monocotyledon leaf. Immunogold labeling was used to detect the cellular and sub-cellular distribution of sucrose synthase (SS) at the electron microscopical level; the protein was visualized using a polyclonal antiserum on embedded tissue sections. Immunolabel was observed in the cytosol of dividing meristematic cells, expanding cells of the elongation zone, and in differentiating cells of young photosynthetic tissue. In fully differentiated leaf tissue, however, the protein was no longer immuno-detectable in photosynthetic cells, but was present in the guard and subsidiary cells of stomata and in companion cells within the phloem tissue of vascular bundles. The tissue- and cell-specific localization of sucrose synthase changes along the growing leaf as a function of the developmental state and the associated need for sucrose import or export.     

Photosynthetic gene expression and cellular differentiation in developing maize leaves

We have exploited the positional gradient of cellular differentiation in Zea mays leaves to study the accumulation of mRNAs encoding subunits of the two CO₂-fixing enzymes and the major chlorophyll-binding protein. These three proteins are differentially compartmentalized in the two photosynthetically active cell types of the leaf. Previous studies have shown that accumulation of the two carboxylases commences 2 to 4 cm from the base of the leaf (Mayfield SP, WC Taylor Planta 161: 481-486) at a position where bundle sheath and mesophyll cells show morphological evidence of maturation. The light-harvesting chlorophyll a/b protein accumulates progressively from the leaf base, as does its mRNA, in spite of its localization in mesophyll cells after cellular differentiation occurs. While small quantities of phosphoenolpyruvate carboxylase mRNA are detectable in the basal region of the leaf, significant mRNA accumulation is coincident with that of the polypeptide at 4 to 6 cm from the leaf base, the region where bundle sheath and mesophyll cells exhibit fully differentiated morphologies. mRNAs encoding the small and large subunits of ribulose 1,5-bisphosphate carboxylase accumulate to significant levels before bundle sheath cells are fully differentiated and before their polypeptides are detectable. Cytological examination indicates that this is the position at which the maturation of intermediate vascular bundles is first evident. Cytosolically localized small subunit mRNA and chloroplast-localized large subunit mRNA are complexed with polyribosomes at all positions of the leaf.     

Lipid biosynthesis by intact mesophyll and bundle sheath chloroplasts from maize

The two dimorphic forms of chloroplast isolated from maize leaves utilized acetate for fatty acid biosynthesis and had similar requirements for cofactors. The oleate:palmitate ratio of the fatty acid products was lower for bundle sheath chloroplasts as was acetate incorporation into total fatty acids. Galactose from UDP-galactose was incorporated into galactolipids by both morphological forms to give monogalactosyl diacylglycerol and digalactosyl diacylglycerol in the ratio of 4:1.     

Sugar uptake and starch biosynthesis by slices of developing maize endosperm

¹⁴C-Sugar uptake and incorporation into starch by slices of developing maize (Zea mays L.) endosperm were examined and compared with sugar uptake by maize endosperm-derived suspension cultures. Rates of sucrose, fructose, and d- and l-glucose uptake by slices were similar, whereas uptake rates for these sugars differed greatly in suspension cultures. Concentration dependence of sucrose, fructose, and d-glucose uptake was biphasic (consisting of linear plus saturable components) with suspension cultures but linear with slices. These and other differences suggest that endosperm slices are freely permeable to sugars. After diffusion into the slices, sugars were metabolized and incorporated into starch. Starch synthesis, but not sugar accumulation, was greatly reduced by 2.5 millimolar p-chloromercuribenzenesulfonic acid and 0.1 millimolar carbonyl cyanide m-chlorophenylhydrazone. Starch synthesis was dependent on kernel age and incubation temperature, but not on external pH (5 through 8). Competing sugars generally did not affect the distribution of ¹⁴C among the soluble sugars extracted from endosperm slices incubated in ¹⁴C-sugars. Competing hexoses reduced the incorporation of ¹⁴C into starch, but competing sucrose did not, suggesting that sucrose is not a necessary intermediate in starch biosynthesis. The bidirectional permeability of endosperm slices to sugars makes the characterization of sugar transport into endosperm slices impossible, however the model system is useful for experiments dealing with starch biosynthesis which occurs in the metabolically active tissue.     

Lipid biosynthesis by chloroplasts isolated from developing Zea mays

Fatty acid biosynthesis by isolated plastids has been examined in relation to chloroplast development and differentiation in leaves of maize plants grown in light for 7 days. Biosynthesis of fatty acids from acetate by proplastids prepared from the basal regions of the leaf was low and mainly palmitate was synthesized. The greatly increased utilization of acetate for fatty acid biosynthesis as the plastids increased in size was due to an increased synthesis of oleate. The maximum synthesis of total fatty acids and monoenoic fatty acids was obtained in chloroplasts prepared from leaf tissue 6–8 cm from the base of the plant where granal formation was most active. Fully-developed chloroplasts prepared from distal regions of the leaf were less active in fatty acid biosynthesis. Maize chloroplasts failed to synthesize fatty acids when isolated by methods commonly used to prepare active spinach chloroplasts. The method of isolation which included a density gradient gave a high proportion of Class I chloroplasts from maize leaves and incorporated up to about 10% of the acetate used. Biosynthesis of unsaturated fatty acids, especially with chloroplasts prepared from the most mature tissue, was increased by the addition of both mitochondrial and microsomal fractions. Increases in polyunsaturated fatty acids were also obtained but the proportions in the newly-synthesized fatty acids were well below the endogenous levels. Monoenoic synthesis was greatly stimulated by increasing the pH in the range 7·0–8·0 and also the highest proportions of unsaturated fatty acids were obtained at short incubation times.     

Differential expression of candidate genes for lignin biosynthesis under drought stress in maize leaves

In order to provide information for the development of molecular selection markers for drought tolerance improvement, the methods of prometric analysis, quantitative real-time PCR and field evaluation were employed for the identification of the differential expression of candidate genes under drought stress in maize. At seventeen, twenty-four and forty-eight hours of polyethylene glycol-simulated drought stress at the seventh leaf stage, leaf samples were collected from two drought-tolerant inbred lines for prometric analysis by two-dimensional electrophoresis and peptide mass fingerprinting. Fifty-eight proteins out of more than 500 were found in response to drought stress. Three drought-induced spots 2506, 3507 and 4506 showed sequence similarity with cinnamyl alcohol dehydrogenase, cytochrome protein 96A8 and S-adenosyl-L-methionine synthase, respectively. The expression of two key enzymes to lignin biosynthesis was quantified by quantitative real-time PCR among three drought-tolerant and one drought-sensitive inbred lines under drought stress and well-watered control conditions. After a decrease at the beginning of drought stress, the expression of cinnamyl alcohol dehydrogenase and caffeateO-methyltransferase recovered at twenty-four hours of the drought stress in the three drought-tolerant lines, but not in the drought-sensitive lines. Leaf lignin content, anthesis-silking interval and grain weight per plant were investigated with six inbred lines of varying drought tolerance under drought stress and well-watered control. Drought tolerance coefficients of these three characters were calculated and the correlation coefficients among these drought tolerance coefficients were estimated. Significant difference in leaf lignin content was found among the inbred lines and in response to drought stress. Close correlations were observed between the drought tolerant coefficients for leaf lignin content and grain weight per plant, and between the drought tolerant coefficients for leaf lignin content and anthesis-silking interval. These results indicate that leaf lignin content is a useful index for evaluation of drought tolerance in maize. Molecular selection markers can be developed on the basis of differential expression of the candidate genes and applied to maize improvement for drought tolerance.     

Nitric oxide mediates brassinosteroid-induced ABA biosynthesis involved in oxidative stress tolerance in maize leaves

The role of ABA in brassinosteroid (BR)-induced stress tolerance and the relationship between BR, nitric oxide (NO) and ABA under water stress induced by polyethylene glycol (PEG) were investigated in leaves of maize (Zea mays) plants. Water stress led to oxidative damage. Pre-treatment with the BR biosynthetic inhibitor brassinazole (Brz) aggravated the oxidative damage induced by PEG treatment, which was alleviated by the application of BR or ABA. Pre-treatment with the ABA biosynthetic inhibitor fluridone also aggravated the oxidative damage induced by PEG treatment; however, this was barely alleviated by the application of BR. BR treatment increased the content of ABA and up-regulated the expression of the ABA biosynthetic gene vp14 in maize leaves, which was blocked by pre-treatments with the NO scavenger cPTIO (2,4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) and the nitric oxide synthase inhibitor l-NAME (N(G)-nitro-l-arginine methyl ester. Moreover, BR treatment induced increases in the generation of NO in mesophyll cells of maize leaves, and treatment with the NO donor sodium nitroprusside (SNP) up-regulated the content of ABA and the expression of vp14 in maize leaves. Our results suggest that BR-induced NO production and NO-activated ABA biosynthesis are important mechanisms for BR-enhanced water stress tolerance in leaves of maize plants.     

Spatial and temporal profiles of cytokinin biosynthesis and accumulation in developing caryopses of maize

Background and Aims

Cytokinins are a major group of plant hormones and are associated with various developmental processes. Developing caryopses of maize have high levels of cytokinins, but little is known about their spatial and temporal distribution. The localization and quantification of cytokinins was investigated in maize (Zea mays) caryopsis from 0 to 28 d after pollination together with the expression and localization of isopentenyltransferase ZmIPT1 involved in cytokinin biosynthesis and ZmCNGT, the gene putatively involved in N9-glucosylation.

Methods

Biochemical, cellular and molecular approaches resolved the overall cytokinin profiles, and several gene expression assays were used for two critical genes to assess cytokinin cell-specific biosynthesis and conversion to the biologically inactive form. Cytokinins were immunolocalized for the first time in maize caryopses.

Key Results

During the period 0–28 d after pollination (DAP): (1) large quantities of cytokinins were detected in the maternal pedicel region relative to the filial tissues during the early stages after fertilization; (2) unpollinated ovules did not accumulate cytokinins; (3) the maternal nucellar region showed little or no cytokinin signal; (4) the highest cytokinin concentrations in filial endosperm and embryo were detected at 12 DAP, predominantly zeatin riboside and zeatin-9-glucoside, respectively; and (5) a strong cytokinin immuno-signal was detected in specific cell types in the pedicel, endosperm and embryo.

Conclusions

The cytokinins of developing maize caryopsis may originate from both local syntheses as well as by transport. High levels of fertilization-dependent cytokinins in the pedicel suggest filial control on metabolism in the maternal tissue; they may also trigger developmental programmed cell death in the pedicel.     

Tissue-distribution of secondary phenolic biosynthesis in developing primary leaves of Avena sativa L.

Primary leaves of oats (Avena sativa L.) have been used to study the integration of secondary phenolic metabolism into organ differentiation and development. In particular, the tissue-specific distribution of products and enzymes involved in their biosynthesis has been investigated. C-Glucosylflavones along with minor amounts of hydroxycinnamic-acid esters constitute the soluble phenolic compounds in these leaves. In addition, considerable amounts of insoluble products such as lignin and wall-bound ferulic-acid esters are formed. The tissue-specific activities of seven enzymes were determined in different stages of leaf growth. The rate-limiting enzyme of flavonoid biosynthesis in this system, chalcone synthase, together with chalcone isomerase (EC 5.5.1.6) and the terminal enzymes of the vitexin and isovitexin branches of the pathway (a flavonoid O-methyltransferase and an isovitexin arabinosyltransferase) are located in the leaf mesophyll. Since the flavonoids accumulate predominantly (up to 70%) in both epidermal layers, an intercellular transport of products is postulated. In contrast to the flavonoid enzymes, L-phenylalanine ammonia-lyase (EC 4.3.1.5), 4-coumarate: CoA ligase (EC 6.2.1.12), and S-adenosyl-L-methionine: caffeate 3-O-methyltransferase (EC 2.1.1.-), all involved in general phenylpropanoid metabolism, showed highest activities in the basal leaf region as well as in the epidermis and the vascular bundles. We suggest that these latter enzymes participate mainly in the biosynthesis of non-flavonoid phenolic products, such as lignin in the xylem tissue and wall-bound hydroxycinnamic acid-esters in epidermal, phloem, and sclerenchyma tissues.Abbreviations CHI chalcone isomerase - CHS chalcone synthase - 4CL 4-coumarate: CoA ligase - CMT S-adenosyl-L-methionine:caffeate 3-O-methyltransferase - FMT S-adenosyl-L-methionine:vitexin 2-O-rhamnoside 7-O-methyltransferase - HPLC high-performance liquid chromatography - IAT uridine 5-diphosphate L-arabinose:isovitexin 2-O-arabinosyltransferase - PAL L-phenylalanine ammonia-lyase     

Auxin biosynthesis in maize

For the biosynthesis of the phytohormone indole-3-acetic acid (IAA), a number of tryptophan-dependent and -independent pathways have been discussed. Maize is an appropriate model system to analyze IAA biosynthesis particularly because high quantities of IAA conjugates are stored in the endosperm. This allowed precursor feeding experiments in a kernel culture system followed by retrobiosynthetic NMR analysis, which strongly suggested that tryptophan-dependent IAA synthesis is the predominant route for auxin biosynthesis in the maize kernel. Two nitrilases ZmNIT1 and ZmNIT2 are expressed in seeds. ZmNIT2 efficiently hydrolyzes indole-3-acetonitrile (IAN) to IAA and thus could be involved in auxin biosynthesis. Redundant pathways, e.g., via indole-3-acetaldehyde could imply that multiple mutants will be necessary to obtain IAA-deficient plants and to conclusively identify relevant genes for IAA biosynthesis.     

Carbon dioxide fixation and related properties in sections of the developing green maize leaf

Light and dark ¹⁴CO₂ assimilation, pulse-chase (¹⁴CO₂ followed by ¹²CO₂) labeling experiments both in the light and in the dark, photorespiratory activity and some enzymes (ribulose 1,5-bisphosphate (RuBP) carboxylase, phosphoenolpyruvate (PEP) carboxylase, and NADP-malic enzyme) were followed in sections of 2.5 centimeters from the base (younger tissue) to the tip (oldest tissue) of the green maize leaf. Tissue was taken from the third leaf of 12- to 16-day-old plants consisting of sections 0 to 2.5 centimeters (base), 4.5 to 7.0 centimeters (center) and 9.0 to 11.5 centimeters (top) measured from the base. Some of these properties were also determined in the intact leaves of 4-day-old maize plants.