Dark fixation of CO2 during gluconeogenesis by the cotyledons of Cucurbita pepo L.

We did this work to discover the pathway of CO₂ fixation into sugars in the dark during gluconeogenesis by the cotyledons of 5-day-old seedlings of Cucurbita pepo L. We paid particular attention to the possibility of a contribution from ribulosebisphosphate carboxylase. The detailed distribution of ¹⁴C after exposure of excised cotyledons to ¹⁴CO₂ in the dark was determined in a series of pulse and chase experiments. After 4s in ¹⁴CO₂, 89% of the ¹⁴C fixed was in malate and aspartate. In longer exposures, and in chases in ¹²CO₂, label appeared in alanine, phosphoenolpyruvate, 3-phosphoglycerate and sugar phosphates, and accumulated in sugars. The transfer of label from C-4 acids to sugars was restricted by inhibition of phosphoenolpyruvate carboxykinase in vivo by 3-mercaptopicolinic acid. We conclude as follows. Initial fixation of CO₂ in the dark is almost entirely into phosphoenolpyruvate, probably via phosphoenolpyruvate carboxylase (EC 4.1.1.31) which we showed to be present in appreciable amounts. Incorporation into sugars occurs chiefly, if not completely, as a result of randomization of the carboxyl groups of the C-4 acids and subsequent conversion of the oxaloacetate to sugars via the accepted sequence for gluconeogenesis. Ribulosebisphosphate carboxylase appears to make very little contribution to sugar synthesis from fat.     

Factors affecting plantlet regeneration from <Emphasis Type="Italic">in vitro</Emphasis> cultured immature embryos and cotyledons of <Emphasis Type="Italic">Prunus mume</Emphasis> “Xue mei”

Using immature embryos and cotyledons as explants, a successful system to culture immature embryos and induce direct regeneration from cotyledons was established for Prunus mume “Xuemei”. For immature embryo culture, a high frequency of plantlet formation (89.5%) from the embryonic axis was obtained using half-strength Murashige and Skoog (1/2 MS) medium supplemented with 13.2 μM 6-benzyladenine (BA) and 2.7 μM 1-naphthaleneacetic (NAA). Shoots formed directly from cotyledons with the embryo axis intact when explants were cultured on 1/2 MS medium containing 2.2 μM BA with different combinations of NAA (2.7, 5.4 μM) and indole-3-butyric acid (IBA) (0, 2.5, 5.0 μM). Better results were achieved when the embryonic axis was removed from the cotyledons and cultured on 1/2 MS medium supplement with 13.2 μM BA, 2.7 μM NAA or 2.2 μM BA, 2.2 μM thidiazuron (TDZ), and 2.7 μM NAA, respectively. Regenerated shoots were successfully rooted on 1/2 MS or Woody Plant medium (WPM) supplemented with 2.5–5.0 μM IBA. The effect of the embryonic axis, BA, and TDZ on cotyledon regeneration was investigated in detail. Rooted plantlets were transferred to soil successfully.     

Changes in photosynthetic capacity and polypeptide patterns during natural senescence and rejuvenation of <Emphasis Type="Italic">Cucurbita pepo</Emphasis> L. (zucchini) cotyledons

Natural senescence of Cucurbita pepo (zucchini) cotyledons was accompanied by a gradual degradation of reserve proteins (globulins) and an intensive decrease in the content of both large subunit (LSU) and small subunit (SSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The net photosynthetic rate, the primary photochemical activity of PSII, estimated by the variable fluorescence (F_v)/maximal fluorescence (F_m) ratio (F_v/F_m) and the actual quantum yield of PSII electron transport in the light-adapted state (ΦPSII) also progressively decreased during natural senescence. In contrast, the fraction of the absorbed light energy, which is not used for photochemistry (LNU) increased with progression of senescence. The decline in the photosynthetic rate started earlier in ontogenesis compared with the down-regulation of the functional activity of PSII, thus suggesting the existence of protective mechanisms which maintain higher efficiency of the photochemical electron transport reactions of photosynthesis compared with the dark reactions of the Calvin cycle during earlier stages of natural senescence. Decapitation of the epicotyl above the senescing cotyledons resulted in full recovery of the polypeptide profile in the rejuvenated cotyledons. In addition, the photosynthetic rate increased reaching values that exceeded those measured in juvenile cotyledons. The photochemical efficiency of PSII also gradually recovered, although it did not reach the maximum values measured in the presenescent cotyledons.     

提高农杆菌基因转化率方法的研究

通过甘蓝型油菜带柄子叶转化过程中转化受体是否进行预培处理、转化前菌液的对数浓度确定以及合适的再生筛选体系的摸索,研究了提高农杆菌基因转化效率的方法。试验结果表明：转化受体经过MD培养基3d的预培处理,用处于对数生长期OD600为0．6～0．8的菌液稀释至所需浸染浓度,浸染所需时间共培后,转入降低卡那霉素浓度的初筛分化培养基中,再经过后期较高浓度的卡那霉素继代筛选培养,大大提高了转化后卡那霉素抗性绿苗的得率。此研究不仅优化了油菜的转化体系和提高了油菜的转化效率,而且为农杆菌转化其它不同物种的受体材料提供了可指导的借鉴。     

莲子叶发育过程的光学和电子显微镜观察

莲(Nelumbo nucifera Gaertn)的子叶从子叶原基发生至成熟休眠,整个生长期约30天,伹受品种、花期、气温和日照的影响,有3—5天的差异。光学和电子显微镜的观察表明,叶内细胞的形态结构在发育过程中变化巨大,子叶发育格局类似双子叶植物。整个发育过程份四个发育阶段:Ⅰ.细胞增殖,形态发生;Ⅱ.细胞液泡化,体积扩大;Ⅲ.细胞处于功能期,大量合成和积累贮藏物质;Ⅳ.脱水收缩,成熟休眠。位于子叶不同部位的叶肉细胞的发育不同步,通过前三个发育阶段所需时间也各不相同。细胞液泡化和进入功能期的位置顺序是从基部到顶部,先外层后内层。受精后25—26天,几乎全部细胞依次进入成熟休眠。     

The control of food mobilisation in seeds of Cucumis sativus L.

The degradation of neutral lipid and the development of lipase activity in cucumber cotyledons is stimulated by white light. Malate synthase and isocitrate lyase activities show no stimulation. Lipase activity and neutral lipid breakdown are also enhanced by red light, far-red light proving ineffective. Far-red light reverses the effect of red light indicating the involvement of phytochrome in the control of lipase activity. Although light stimulates neutral lipid degradation it appears that much of the additional lipid lost is used in the synthesis of polar lipid constituents. Furthermore, the influence of light on lipid degradation appears to be species dependent.Abbreviations WL white light - RL red light - FRL far-red light     

Structural analysis of colonies derived from sunflower (Helianthus annuus L.) protoplasts cultured in liquid and semi-solid media

Summary Colonies have been generated from cotyledon protoplasts of sunflower (Helianthus annuus L.) and transferred to solid regeneration medium. The development of these protoplasts was strikingly dependent on their physical environment (liquid or solidified medium) and the nature of the gelling agent (agarose, alginate). In particular, extremely compact multicellular structures developed in agarose solidified medium, similar to those observed in cultures of hypocotyl- and petiole-derived sunflower protoplasts (termed proembryoids). A detailed study at the histological level, however, indicates that these structures are degenerative, rather than embryonic in nature. The quality of the colonies, with regard to their regeneration potential, appears to be best in alginate solidified medium.Abbreviations MS medium of Murashige and Skoog (1962) - NAA 1-naphthaleneacetic acid - BAP 6-benzylaminopurine     

一种鉴定金花茶人工杂交F_1代杂种苗的简易方法

本试验选用了两个以金花茶作父本的人工杂交组合F_1代实生苗,经根尖体细胞染色体观察鉴定四倍体杂种。一、云南野山茶(Camellia pitardii var.yunnanica六倍体)×金花茶(C.Chrysantha二倍体)。结果为:所获56株子叶和下胚轴为红色的F_1代杂种苗中,有55株(98.2％)是真正的四倍体杂种(X~2=0.0128,D.f.=1,0.95>p>0.90);所获50株子叶和下胚轴为黄白色的杂种苗中,有13株(26％)为真正四倍体杂种(X~2=27.38,D.f.=13,0.02>p>0.01)。二、云南山茶花(C.reticulata六倍体)×金花茶。结果为:在78株子叶和下胚轴为红色的杂交苗中,有77株(98.7％)为真正四倍体杂种(X~2=0.0128,D.f.=1,0.95>p>0.90);而在78株子叶及下胚轴为黄白色的杂种苗中,只有21株(26.9％)是真正四倍体杂种(X~2=41.65,D.f.=24,0.02>p>0.01) 在多数杂种实生苗中的这种红色素,是因种子直感现象而发生的父本金花茶的一种遗传性状。金花茶的另一特征——多子叶现象(3枚以上),则在一些F_1代杂种苗或杂种种子中表现得不明显。因此,利用F_1代杂种苗子叶和下胚轴所具有的红色特征,在杂种种子萌发期用来鉴别以金花茶为亲本的F_1代杂种的真伪,是一种简便、快速的、有发展前途的方法。     

Characterization and subcellular localization of vicilin and phytohemagglutinin,the two major reserve proteins of Phaseolus vulgaris L.

Extracts of bean (Phaseolus vulgaris L. cv. Greensleeves) cotyledons contained two abundant proteins: vicilin and phytohemagglutinin. Vicilin, a 6.9 S protein fraction at neutral pH, associated to an 18.0 S form at pH 4.5 and had 3 non-identical subunits with molecular weights (MW) of 52,000, 49,000 and 46,000. Phytohemagglutinin, a 6.4 S protein fraction, had 2 non-identical subunits with MW of 34,000 and 36,000. Phytohemagglutinin could be separated by isoelectrofocusing into a mitogenic and non-erythroagglutinating protein with a single subunit of MW=34,000, and a mitogenic and erythroagglutinating protein fraction which contained both subunits. Vicilin is apparently identical with the so called glycoprotein II (A. Pusztai and W.B. Watt, Biochim. Biophys. Acta 365, 57–71, 1970) and with globulin G1 (R.C. McLeester, T.C. Hall, S.M. Sun, F.A. Bliss, Phytochem. 2, 85; 1973), while phytohemagglutinin is identical with globulin G2 (McLeester et al., 1973). Since vicilin and phytohemagglutinin are internationally used names there is no need to introduce new names to describe P. vulgaris reserve proteins. Both proteins are catabolized in the course of seedling growth and are located in the protein bodies, indicating that they are reserve proteins. Vicilin isolated in its 18.0 S form from the cotyledons of young seedlings contains substantial quantities of smaller polypeptides, in addition the 3 original ones. We suggest that the presence of these small polypeptides represents partial breakdown of the vicilin prior to its complete catabolism.     

Development and intracellular localization of lipase activity in rapessed (Brassica napus L.) cotyledons

In homogenates of resting rapeseeds no lipase activity (glycerolester hydrolase, EC 3.1.1.3) could be detected using a titrimetric assay procedure. Following a 30-h lag-phase after imbibition, lipase activity increased sharply, reaching its maximum at day 4 after sowing. Simultaneously triglyceride content of the cotyledons decreased sharply. At any time during the 11-day period of seedling growth examined, only an alkaline lipase activity with a pH optimum around 9 was present. White light had essentially no effect on the development of lipase activity. However, the disappearance of lipase activity from the cotyledons after fat utilization was found to depend on nitrogen nutrition of the seedlings. The activities of the glyoxysomal enzymes catalase and malate synthetase showed the usual rise and fall patterns with peak activities at day 4 after sowing, independently of the mineral nutrition of the seedlings.About 90% of the lipase activity was associated with a microsomal membrane fraction. Resolution of this fraction by sucrose density gradient centrifugation (62,000 g for 14 h) yielded three distinct membrane fractions. Maximum activities of membrane marker enzymes were recovered from the gradients at following densities: The major portion of microsomal protein and lipase activity at 1.085 kg/l; microsomal malate synthetase and phosphorylcholineglyceride transferase at 1.116 kg/l; NADH-cytochrome c reductase and phosphorylcholinecytidyl transferase at 1.133 kg/l. Evidently in rapeseed cotyledons lipase activity is associated only with a discrete microsomal membrane fraction which sediments differently from membrane fractions of the endoplasmic reticulum.