Quantitative aspects of latex lipid synthesis in Euphoribia lathyris L. seedlings

Changes in the dry weight of the endosperm of Euphorbia lathyris L. seedlings showed that 2 mg material was taken up by the cotyledons after 10 d germination. A similar amount of sucrose could be taken up by these seedlings after removal of the endosperm. The maximum yield of latex triterpenes synthesized from this exogenously supplied substrate was in the same order of magnitude as the daily latex lipid increase in 19 g per seedling. Cotyledons and adjacent 1–2 cm segment of the hypocotyl were the most active tissues in latex trieterpene synthesis. Excised cotyledons were able to accumulate 1–1.5 mg sucrose in 48 h from a sugar concentration higher than 0.1 mol l^-1. In this period a maximum amount of 8–10 g latex triterpenes could be synthesized from this substrate. [¹⁴C]Mevalonic acid was rapidly taken up by excised cotyledons but not metabolized by the laticifers. This exogenously supplied precursor was rapidly converted to squalene and triterpenes by the adjacent tissue, and after 48 h incubation most of the ¹⁴C in the nonsaponifiable fraction was traced in the phytosterolds.     

Accumulation of non-utilizable starch in laticifers of Euphorbia heterophylla and E. myrsinites

Starch biosynthesis and degradation was studied in seedlings and mature plants of Euphorbia heterophylla L. and E. myrsinites L. Mature embryos, which lack starch grains in the non-articulated laticifers, develop into seedlings that accumulate starch rapidly when grown either in the light or the dark. Starch accumulation in laticifers of dark-grown seedlings was ca. 47 and 43% of total starch in light-grown controls in E. heterophylla and E. myrsinites, respectively. In light-grown seedlings, starch was present in laticifers as well as parenchyma of stems and leaves, whereas in dark-grown seedlings starch synthesis was almost exclusively limited to laticifers. In 7-month-old plants placed into total darkness, the starch in chyma was depleted within 6 d, whereas starch in laticifers was not mobilized. The starch content of latex in plants during development of floral primordia, flowering, and subsequent fruit formation remained rather constant. The results indicate that laticifers in seedlings divert embryonal storage reserves to synthesize starch even under stress conditions (darkness) in contrast to other cells, and that starch accumulated in laticifers does not serve as a metabolic reserve. The laticifer in Euphorbia functions in the accumulation and storage of secondary metabolites yet retains the capacity to produce, but not utilize starch, a primary metabolite.     

Biosynthesis of pentacyclic triterpenoids in leaves of Ilex aquifolium L.

Ilex leaves can utilize sucrose and mevalonate for the synthesis of triterpene esters. Mevalonate is also used for the synthesis of free triterpenoid alcohols, but sucrose is not. The selectivity of precursor utilization indicates separate sites for triterpenol and triterpenol-ester synthesis. The sites of synthesis are not found at the main locations of accumulation of triterpenols (the epicuticular wax) or triterpene esters (the cytoplasmatic lipid globules). Transport from the site of synthesis to the lipid globules, and especially towards the epicuticular wax, is slow.Abbreviations MVA mevalonic acid lactone - TLC thin layer chromatography     

Bioactive triterpene derivatives from latex of two Euphorbia species

We have investigated the antifeedant and toxic effects of 23 semisynthetic terpenoid derivatives obtained through chemical modifications of the major components of Euphorbia resinifera (alpha-euphol and alpha-euphorbol) and E. officinarum (obtusifoliol and 31-norlanostenol) latex on several insect species (Spodoptera littoralis, Myzus persicae and Rhopalosiphum padi), their selective cytotoxicity on insect Sf9 and mammalian CHO cells and their phytotoxic effects on Lactuca sativa. The conversions focused mainly on positions 3,7,11, and 24 with several oxidizing agents. A total of 18 compounds affected S. littoralis growth (IGR). Our results support the importance of the C-3 substituent, suggest the involvement of the C-7 substituent and indicate that the C-3 hydroxyl is not essential for the IGR effect. Overall, Sf9 cells were more sensitive to the active compounds than CHO cells. All of these compounds had non selective moderate phytotoxic effects on radicle elongation of L. sativa.     

Phylogeny of subsect. Meleuphorbia (A. Berger) Pax & Hoffm. (Euphorbia L.) reflects the climatic regime in South Africa

The nuclear ITS region and four noncoding cpDNA regions were directly sequenced to reconstruct the phylogeny of subsect. Meleuphorbia (Euphorbia L.), which is endemic to South Africa. Sequence polymorphism within cpDNA regions was too low to permit phylogenetic analyses. Large deletions including the ctp2-atpB proximal promoter in the atpB-rbcL IGS were found in two individuals. Phylogeny of subsect. Meleuphorbia was reconstructed using nrITS sequence data. The two resulting major clades are consistent with the geographic distribution of the investigated taxa. Plants distributed in the winter rainfall area in the Little Karoo form one clade, plants growing in the Great Karoo with predominant summer rainfall form the other. Subsect. Meleuphorbia is not monophyletic, because members of subsect. Anthacantha are clustered within the meleuphorbias. Both share functional unisexuality, form angled stems and the key character of Anthacantha (inflorescence spines) occurs as a transitional stage in form of persistent peduncles in Meleuphorbia.     

Cytochemical localization of pectinase activity in laticifers ofNerium oleander L.

Summary A method is described for the cytochemical localization of pectinase activity at the ultrastructural level. The procedure involves the use of Benedict's reagent to form an electron-dense copper precipitate when reacted with reducing sugars liberated from exogenously supplied pectin. Using this technique, pectinase activity was examined in the nonarticulated, branched laticifers ofNerium oleander. Electron opaque crystalline deposits indicating the presence of pectolytic enzymes were identified in laticifer central vacuoles. Smaller amounts of reaction product were distributed along the middle lamella between laticifers and adjacent cells. This report represents the first direct evidence for the involvement of pectinase in intrusive growth of nonarticulated laticifers.     

Hormonal effects on phyllotaxis ofEuphorbia lathyris L.

Benzyladenine and gibberellic acid sprays (100 mg/l) onEuphorbia lathyris L. plants were absolutely successful (100% of the plants) in inducing a change in its normal decussate phyllotaxis. Benzyladenine produced a change to tetracussate, tricussate and bijugate, and gibberellic acid to spiral phyllotaxis. Benzyladenine and gibberellic acid treatments resulted in a significant increase in apex diameter (72.8% and 19.1% respectively). CCC, Ancymidol, Alar and Glyphosine did not alter decussate phyllotaxis.     

Unsaponifiable Lipid Biosynthesis in the Seedling of Euphorbia lathyris L.: A Bypass via Alcoholic Fermentation

[1-¹⁴C]-ethanol supplied to the cotyledons of 9-d-old Euphorbialathyris seedlings was rapidly incorporated into unsaponifiablelipids, particularly into sterols, latex triterpenols and intothe triterpene ketones of the epicuticular wax. The [¹⁴C]-triterpenoidproduction from ethanol was hardly affected by sucrose in theexternal medium when sucrose uptake rates were low, but whenthe uptake rate was higher the [¹⁴C]-triterpenoid productionfrom [¹⁴C]-ethanol was greatly reduced. This observation isconsistent with the proposition that at high sucrose uptakerates, some sucrose is converted into ethanol, so that the incorporationof [¹⁴C]-ethanol into triterpenoids is reduced by competitionwith endogenously formed ethanol. A calculation based on theputative daily ethanol production in the cotyledons and thedaily triterpenoid production of seedlings indicates that about10 % of the triterpenoid synthesis in vivo may be from ethanol. Ethanol, Euphorbia lathyris, fermentation, seedling, triterpenoid biosynthesis     

Trenching Behavior by Caterpillars of the Euphorbia Specialist, Pygarctia roseicapitis: A Field Study

Six-hour continuous observations on the arctiid caterpillar, Pygarctia roseicapitis, were made under natural conditions in the field, and all foraging-related activities were recorded. Individuals invariably cut veins of the latex-rich host plants before feeding distal to the cuts. Seven percent of the time was spent in this vein-cutting behavior, and 40% of the time was spent feeding. Most vein-cutting events were on petioles, allowing up to six feeding bouts on the resultant wilted leaf. Both leaves and plants were often abandoned before the resource was fully utilized. The results are discussed in relation to vein-cutting behavior by other insect herbivores and the possible trade-offs in such strategies.     

续随子的组织培养与快速繁殖

本文对能源植物续随子（Euphorbia lathyris L．）进行离体培养与快速繁殖技术研究。结果表明：以种子、茎段和顶芽为外植体均能诱导获得无菌苗,其中以茎段或顶芽的芽诱导率较高;幼芽继代增殖的最佳培养基是White＋TDZ0．05mg·L。＋IBA0．01mg·L^（-1）＋0．1％活性炭,培养25d的增殖系数约为4．5;而最适生根培养基为1／2MS＋NAA1．0mg．L^（-1）＋0．1％活性炭,炼苗移栽后,成活率约为60％。培养过程中,通过控制细胞分裂素浓度能有效解决续随子培养中的玻璃化问题,添加0．1％的活性炭对缓解外植体和无菌苗褐化效果较好。     

Lysosome and proteasome pathways are distributed in laticifers of Euphorbia helioscopia L.

At present, the lysosome pathway (LP) and proteasome pathway (PP) are known as major clearance systems in eukaryotic cells. The laticifer, a secretory tissue, degrades some cytoplasm during development. In this study, we investigated the distribution of LP and PP in non‐articulated laticifers of Euphorbia helioscopia L. Electron microscopy revealed that, plastids, mitochondria and some cyotsol were degraded in the late development laticifers, where there were numerous vesicles originated from dicytosomes. Accordingly, some key proteins in LP and PP were detected in E. helioscopia latex using isobaric tags for relative and absolute quantitation (iTRAQ) proteomics. Further immunohistochemistry analysis revealed that the clathrin heavy chain (CHC) belonging to LP and the ubiquitin‐mediated proteasome degradation increases gradually as the laticifer develops. Immuno‐electron microscopy revealed that the cysteine protease, CHC and AP‐2 complex subunit beta‐1 belonging to LP were mainly distributed in vesicles deriving from dicytosomes, which we called lysosome‐like vesicles. Ubiquitin was widely distributed in the cytosol, and proteasome activity was significantly reduced when various concentrations of the inhibitor MG132 were added to the latex total protein. We hypothesize that LP and PP are distributed in E. helioscopia laticifers; and it was speculated that LP and PP might be involved in the degradation of organelles and some cytoplasmic matrix in E. helioscopia laticifers.     

Chromoplasts of Tropaeolum majus L.: Lipid synthesis in whole organelles and subfractions

Isolation of tubulous chromoplasts from Tropaeolum majus L. petals was achieved in pure form. Their main substructures-lipid bodies, tubules, and envelope membranes-have been enriched. Whole chromoplasts as well as substructures have been tested for their activities in lipid synthesis. The following activities were found: fatty acid synthesis from acetate, glycosyl transfer reactions from UDP-galactose and UDP-glucose to galactolipids and sterols, acyltransferase reactions from palmitoyl-CoA, and a very active acyl-CoA hydrolase (EC 3.1.2.2.). Fatty acid synthesis was restricted to whole chromoplasts. Glycosyl- and acyltransferases were essentially confined to envelope membranes, whereas acyl-CoA hydrolase was found in all fractions. The chemical composition of chromoplast subfractions was determined. The lipid bodies consisted mainly of galactolipids and carotenoid esters in a 1:1 ratio, together with small amounts of protein.     

二倍体与四倍体绿玉树乳汁管分布特征的比较

采用石蜡切片法、离析法以及徒手切片3种方法研究了绿玉树(EuphorbiatirucalliL.)乳汁管的分布,并对二倍体和四倍体植株茎的乳汁管分布和数量进行了比较研究。结果表明,绿玉树乳汁管为无节型;初级乳汁管主要分布在木质部与韧皮部之间,在2a生茎的髓部也有分布;次级乳汁管以多级分支的形式无规则地散布在韧皮部中;末级乳汁管分布在皮层(或周皮)下部。随着茎成熟度增加,初级乳汁管数量和直径随之增加,直径增加到一定时便不再增粗,而以增加数量的方式来缓解液压。与二倍体相比,相同成熟度的四倍体绿玉树乳汁管的数量和直径都显著减少,其中嫩茎的初级乳汁管数量减少了5.5条,直径减少了8.4μm;1a生枝条初级乳汁管数量减少了10.7条,直径减少了9.6μm。     

Carotenoid synthesis and pleiotropic effects in carotenoid-deficient seedlings of maize

Plastid-envelope membranes from seedlings ofZea mays L. made carotenoid-deficient by either norflurazon treatment or mutation lack an activity permitting conversion of phytoene to -carotene. This activity in membrane fractions was measured by coincubation in vitro with a soluble system from spinach chloroplasts capable of converting [¹⁴C]isopentenyl pyrophosphate into phytoene. When grown in light, the carotenoid-deficient seedlings lack many soluble chloroplast proteins, including NADP-dependent malic enzyme (EC 1.1.1.40), pyruvate phosphate dikinase (EC 2.7.9.1), and ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39), but apparently still contain the soluble activities permitting synthesis of phytoene.Abbreviations IPP isopentenyl pyrophosphate - LHCP light-harvesting chlorophylla/b-binding protein - norflurazon 4-chloro-5(methylamine)-2-(,,-trifluoro-m-tolyl)-3-(2H)-pyrazinone - TLC thin-layer chromatography - Tris 3-amino-2-(dihydroxymethyl)-1,3-propanediol     

Immunocytochemical identification of laticifers

Summary sensitive immunocytochemical method for the identification of laticifers has been developed. Frozen sections of various laticifer-bearing plant material, mounted on slides, were first flooded with the IgG fraction of rabbit anti-latex antiserum, prepared using whole latex ofAsclepias syriaca, then flooded with fluorescein-conjugated IgG fraction goat anti-rabbit IgG to visualize laticifers. Positive fluorescence was observed for laticifers in shoots and embryos ofA. syriaca andStapelia bella and embryos ofA. tuberosa. Laticifers did not fluoresce in shoots ofA. tuberosa andEuphorbia tirucalli, in embryos ofE. marginata, or in petioles ofMusa paradisiaca andCichorium intybus. Controls prepared with uninjected rabbit serum were negative (no fluorescence).     

Jatrophane diterpenes from Euphorbia spp. as modulators of multidrug resistance in cancer therapy

A phytochemical investigation of the aerial parts of Euphorbia spp., considered one of the most common elements of Mediterranean landascape, led to the isolation of a large number of bioactive plant metabolites, belonging to the diterpenes family. Above all, over seventy jatrophane, modified jatrophane, segetane, pepluane, and paraliane diterpenoids, fifty of them reported for the first time, were extracted, purified and characterized from Euphorbia dendroides, Euphorbia characias, Euphorbia peplus, Euphorbia paralias and Euphorbia helioscopia. These compounds showed interesting pharmacological activities. In particular, jatrophanes, modified jatrophanes and lathyranes exhibited a potent inhibitory activity against P-glycoprotein (Pgp), a membrane protein that confers cellular ability to resist lethal doses of certain cytotoxic drugs by pumping them to the outside, thus resulting in a reduced cytotoxicity. Among the others, our chemical survey led to isolation of the most powerful inhibitors of daunomycin-efflux activity isolated to date for this class of inhibitors, named euphodendroidin D and pepluanin A. Their efficiency was found to be at least two-fold higher than the conventional modulator cyclosporin A, taken as a reference. In addition, the isolation of a high number of natural structurally-related analogues allowed us to perform Structure Activity Relationship (SAR) studies, without any chemical modification, which gave information on the key pharmacophoric elements of these new class of promising drugs. A further set of diterpene analogues, very recently isolated from sun spurge, E. helioscopia, individually investigated for their Pgp- and BCRP-inhibiting properties, appeared to be specific inhibitors of Pgp since they showed no significant activity against BCRP, thus resembling to the third-generation class of specific MDR inhibitors.     

Intercellular compartmentation of sucrose synthesis in leaves of Zea mays L.

The incorporation of ¹⁴C into sucrose and hexose phosphates during steady-state photosynthesis was examined in intact leaves of Zea mays L. plants. The compartmentation of sucrose synthesis between the bundle sheath and mesophyll cells was determined by the rapid fractionation of the mesophyll and comparison of the labelled sucrose in this compartment with that in a complete leaf after homogenisation. From these experiments it was concluded that the majority of sucrose synthesis occurred in the mesophyll cell type (almost 100% when the time-course of sucrose synthesis was extrapolated to the time of ¹⁴C-pulsing). The distribution of enzymes involved in sucrose synthesis between the two cell types indicated that sucrose-phosphate synthetase was predominantly located in the mesophyll, as was cytosolic (neutral) fructose-1,6-bisphosphatase activity. Stromal (alkaline) fructose-1,6-bisphosphatase activity was found almost exclusively in the bundle-sheath cells. No starch was found in the mesophyll tissue. These data indicate that in Zea mays starch and sucrose synthesis are spatially, separated with sucrose synthesis occurring in the mesophyll compartment and starch synthesis in the bundle sheath.     

1,8-Cineole inhibits root growth and DNA synthesis in the root apical meristem ofBrassica campestris L.

1,8-cineole is a volatile growth inhibitor produced bySalvia species. We examined the effect of this allelopathic compound on the growth of other plants usingBrassica campestris as the test plant. Cineole inhibited germination and growth ofB. campestris in a dosedependent manner. WhenB. campestris was grown for 5 days with various concentrations of cineole, the length of the roots was found to be shorter as the concentration of cineole increased, whereas the length of the hypocotyl remained constant up to 400 μM cineole, indicating that cineole specifically inhibited growth of the root. The mitotic index in the root apical meristem of 3-day-old seedlings decreased from 5.6% to 1.6% when exposed to 400 μM cineole, showing that cineole inhibits the proliferation of root cells. We then examined the effect of cineole on DNA synthesis by indirect immunofluorescence microscopy using antibody raised against 5-bromo-2′-deoxyuridine (BrdU, an analogue of thymidine) in thin sections of samples embedded in Technovit 7100 resin. The results clearly demonstrated that cineole inhibits DNA synthesis in both cell nuclei and organelles in root apical meristem, suggesting that cineole may interfere with the growth of other plant species by inhibiting DNA synthesis in the root apical meristem.     

RNA and protein synthesis in sperm cells isolated from Zea mays L. pollen

Summary Sperm cells are thought to be quiescent in pollen and activated upon pollen germination. To test this hypothesis, protein, RNA and DNA synthesis were assessed in Zea mays sperm cells at different times after isolation from pollen. Protein synthesis changed with time; while some proteins were found to be constitutive in both 0 and 24 h cells, others were synthesized and some disappeared. Overall, the number of proteins detected at 24 h doubled compared with freshly isolated cells. Incorporation of [³H]leucine in 24 h cells was about 50 times that in freshly isolated cells, and that of [5, 6-³H]uridine, about 7 times. Very low incorporation of [6-³H]thymidine into the cells was detected; there was no difference between freshly isolated and 24 h cells. It is possible that the differences in synthetic activity between freshly isolated and 24-h-old cells might correspond to sperm cell activation during pollen tube growth. If so, these metabolic changes may play an important role in fertilization.Supported by funds from a Strategic Grant (D.D.C.) and an Operating Grant (D.J.G.) from the Natural Sciences and Engineering Research Council of Canada