The orientation of primary cilia during the wound response in 3Y1 cells

Summary— The behavior of the primary cilia of 3Y1 cells in the interphase was investigated by indirect immunofluorescence microscopy and transmission electron microscopy, using an antibody for tubulin. At 4.5 h after scraping a part of a confluent cell sheet, the primary cilia of cells facing the wound were located predominantly forward of the nucleus on the wounded side, and were oriented in the direction of the leading lamellae. Cytoplasmic microtubules (MTs), emanating from around the base of the cilia, were well developed in the leading lamellae on the wounded side. On the other hand, in the cells of an unperturbed area away from the wounded edge, the primary cilia remained randomly distributed near the nucleus. The position and a certain well-defined orientation of a pair of centrioles seem to play an important role for the development of cytoplasmic MTs, and consequently the orientation of the centrioles is controlled by the primary cilia.     

Chloroplast biogenesis 76. Regulation of 4-vinyl reduction during conversion of divinyl Mg-protoporphyrin IX to monovinyl protochlorophyllide a is controlled by plastid membrane and stromal factors

Most of the chlorophyll (Chl) a of green plants is formed via two biosynthetic routes, namely the carboxylic divinyl and monovinyl chlorophyll biosynthetic routes. These two routes are linked by (4-vinyl) reductases that convert divinyl tetrapyrroles to monovinyl tetrapyrroles by reduction of the vinyl group at position four of the macrocycle to ethyl. The activities of these two routes are very sensitive to cell disruption. For example in barley leaves, cell disruption, a mandatory step during plastid isolation, results in partial inactivation of the carboxylic divinyl route. Investigations with subplastidic fractions revealed that the carboxylic divinyl and monovinyl biosynthetic routes were regulated by a delicate interaction that involved plastid membranes, stroma, and reduced pyridine nucleotides. While the monovinyl biosynthetic route was very active in isolated plastid membranes, activation of the divinyl biosynthetic route required the joint presence of plastid membranes and stroma. Contrary to expectation, activity of the carboxylic divinyl biosynthetic route was greatly enhanced by addition of NADPH to the lysing buffer used during plastid membranes and stroma preparation. NADPH in cooperation with the plastid stroma may play an important regulatory role during the biosynthesis of divinyl and monovinyl protochlorophyllide a. This revised version was published online in June 2006 with corrections to the Cover Date.     

Growth inhibitors in oat grains. I. Leakage of inhibiting factors from oat grains during imbibition and the effect of these on germination and growth

Spikes of barley ( Hordeum vulgare L.) cultivar Bomi and high-lysine mutants Riso 1508 and Riso 56 were cultured on liquid media at varying N and sucrose levels. Bomi accumulated N in response to increasing N levels in the medium and a higher level was reached than in spikes of intact plants. The distribution of N in salt-soluble, hordein, and non-protein N fractions appeared to be normal. Endosperm dry weight and starch were lower than in intact plants and declined at higher N levels. A linear relationship was observed between starch content and the concentration of sucrose in the endosperm water. Uptake of culture medium by the spikes was affected by both N and sucrose concentration. The mutants had lower dry weights and starch contents, and higher sucrose contents than Bomi. At high N levels, the mutants accumulated less hordein, and more non-protein N than Bomi.     

Transport-related amino acid metabolism in germinating barley grains

When eight [¹⁴C]-labelled amino acids were separately injected into the endosperm of germinating (4 days at 20°C) barley (Hordeum vulgare L. cv. Himalaya) grains, the label was rapidly taken up by the scutellum and further transported to the shoot and roots. Some of the amino acids (leucine, lysine and asparagine) were transported in an intact form through the scutellum to the seedling, whilst glutamic acid and aspartic acid were largely converted to glutamine in the scutellum. Proline was mainly transported unchanged, but a small part of the label appeared in glutamine. Arginine was mostly broken down in the scutellum, possibly providing ammonia for the synthesis of glutamine. During further transport in the seedling there was a partial transfer of label from glutamine to asparagine, particularly in the shoot. None of the amino acids used supplied carbon for the synthesis of sucrose, glucose or fructose. Glutamine synthetase activity was particularly high in the scutellum during the period of rapid amino acid transport.     

Apparent transinhibition of peptide uptake in the scutellum of barley grain

The uptake of glycylsarcosine (Gly-Sar) into scutella separated from germinating grains of barley ( Hordeum vulgare L. cv. Himalaya) is inhibited by other peptides; in most cases the inhibition is not purely competitive but of a mixed type (simultaneous increase in the apparent K_m and decrease in V_max) (Sopanen, T. 1979. FEBS Lett. 108: 447–450). The aim of the present experiments was to elucidate the mechanism of the mixed inhibition by studying how peptides already taken up into the cells affect the uptake of Gly-Sar.
When scutella were preincubated in the presence of various peptides, 11 of the 13 peptides tested inhibited the subsequent uptake of Gly-Sar by 10 to 45%. The inhibition, studied in detail with leucylleucine and prolylproline, was due to a decrease in V_max. The two peptides having no effect were glycylglycine and D-alanyl-L-alanine which are the only peptides known to date acting as purely competitive inhibitors when present together with the substrate Gly-Sar.
Preincubation with leucine, proline and alanine was not inhibitory, although preincubation with the corresponding dipeptides was. This result, together with the demonstration of intact leucylleucine in the scutella after preincubation with leucylleucine, indicates that the inhibition was caused by the intact peptides.
The results support the notion that in the mixed type inhibition the increase in the apparent K_m is due to competition for the carrier at the outside of the membrane, while the decrease in V_max is due to peptides taken up and binding to the carrier at the inside of the membrane.     

Dormancy breaking and frost resistance induction in apple embryos as related to changes in reserve and polar lipids

Detailed dose-response curves were obtained for cytokinin-induced retention of chlorophyll in detached leaves of barley, Hordeum vulgare L. cv. Møyar, over a wide range, 10⁻¹⁰– 2.5 × 10⁻⁴ M , of external concentrations of benzyladeninc or kinetin. The over-all shape of the curves was log-linear and the response was subsensitive. Benzyladenine was somewhat more effective than kinetin. In the curves there were pronounced local minima and maxima both at low, intermediate (10⁻⁷– 10⁻⁵ M ) and high concentrations. At low and high concentrations these deviations from the best smooth curves were reproducible, statistically significant and occurred at the same concentrations for benzyladenine and kinetin. In the intermediate range the deviations from the log-linear line were marked and statistically highly significant in some but not all experiments. The cause of the complexities in the dose response curve is not known, but multiphasic uptake of the cytokinins may be involved.     

Location of sugars in barley seeds during germination by NMR microscopy

The distribution and fluctuation of sugars in germinating barley seeds were examined by ¹³C nuclear magnetic resonance (NMR) spectroscopy, ¹H-NMR imaging and ¹H-NMR localized spectroscopy in relation to morphology. Maltose, sucrose, fructose and oils were detected in intact imbibed seeds by ¹³C-NMR spectra. During the first 6 d of germination, the maltose content increased and the oil content gradually decreased, whilst the levels of sucrose and fructose remained constant. Sugars were located by ¹H-NMR images and ¹H-NMR localized spectra in the vascular bundle of the seeds as well as in the solubilized endosperm. They were also detected in the shoots. The sugars detected in an 80% ethanol shoot extract were sucrose and glucose, which were located in the vascular bundles but not in the mesophyll cells of the coleoptile. They were also located in the basal part of the shoot, but not above 7 mm from the scutellum. The data suggest that the sugars are primarily transported through the vascular bundles and, at the same time, rapidly incorporated into mesophyll cells in the leaves.     

Mild phosphorus stress in barley and a related low-phosphorus-adapted barleygrass: Phosphorus fractions and phosphate absorption in relation to growth

Barleygrass ( Hordeum leporinum ) from Australian low-P (phosphorus) soils and commercial barley ( H. vulgare ) with high fertilizer requirements were grown in solution culture at 3 levels of P supply. The high-P-adapted barley produced more biomass at all levels of P supply and was more responsive to added P in terms of rate of tillering, rate of leaf production, final leaf size, and therefore total shoot weight compared to barleygrass. In both species root: shoot ratio decreased in response to improved tissue P status, even at P levels where total biomass did not respond to P supply. Removal of endosperm reserves of barley reduced total biomass to a greater extent than it altered phosphate absorption rate, thus increasing tissue P status and making plants less responsive to added P. Similarly, barleygrass had a slower growth rate but a comparable P absorption rate to that of barley. Thus barleygrass also accumulated tissue P and was unresponsive to added P. All phosphorus chemical fractions increased in response to improved tissue P status, but to differing extents (inorganic-P > nucleic acid-P > lipid-P > ester-P), suggesting that all P fractions (particularly inorganic P) serve, in part, a storage function. Both barleygrass and barley without endosperm had higher concentrations of all P fractions (particularly inorganic P) than did unaltered barley, but this was due entirely to their higher P status (due to slow growth) rather than to any major difference in P metabolism between species. We conclude that slow growth is more important than interspecific differences in P metabolism, P absorption, or efficiency of P utilization in explaining the success of barleygrass and other low-P-adapted species on infertile soils.     

Endogenous nitrate loss as an assay for nitrate reduction in vivo

An in vivo assay method for nitrate reduction is proposed, based on the use of endogenous nitrate rather than on the accumulation of nitrite. Loss of endogenous nitrate and accumulation of nitrite were studied in barley (Hordeum vulgare L. cv. Gars Clipper ex Napier) leaves. Leaf sections were incubated in the dark in a gaseous environment of air or N₂. Nitrate disappeared under both conditions, the highest loss being observed in tissue under anaerobiosis. Nitrite accumulated only in leaf sections under anaerobiosis, but the amount of nitrite accumulated was much lower than the amount of nitrate lost. A comparative study of the capacity of barley leaf sections to use endogenous nitrate and accumulate nitrite showed that both activities were dependent on temperature in a manner characteristic of enzymatic reactions. Disappearance of endogenous nitrate increased with increasing levels of nitrate in the tissue.     

化学诱变剂和^60Co处理对大麦小孢子低氮胁迫培养的影响

以大麦品种‘花30’为材料,用不同剂量化学诱变剂（EMs和PYM）处理大麦游离小孢子,不同剂量^60Co辐照处理大麦离体穗和干种子,比较其对在低氮胁迫下小孢子培养诱导愈伤组织产量和绿苗分化数量的影响。结果表明,EMS处理离体小孢子和^60Co辐照干种子明显比PYM处理小孢子和^60Co辐照离体穗的培养效果好。     

大麦铁蛋白基因(HvFer1)cDNA的克隆和表达

根据玉米铁蛋白ZmFer1的氨基酸序列,采用同源序列法进行序列拼接和引物设计,RT-PCR扩增获得了1个源自二叶期大麦叶片mRNA的大麦铁蛋白基因cDNA片段,HvFer1(GenBank登录号为EF440353)。HvFer1全长1023 bp,5′非翻译区56 bp,3′非翻译区202 bp,开放阅读框编码254个氨基酸。序列分析表明,此蛋白与已知植物铁蛋白高度同源,相似性介于56.7%～83.7%之间,N端具27个氨基酸的信号肽,C端(84～247)具有1个类似铁蛋白的功能域。RT-PCR表达谱分析显示,HvFer1在大麦的茎、叶、幼穗和幼根均能表达,茎和幼穗中表达量略高些。此外,HvFer1受PEG和盐胁迫的强烈诱导表达,中度铁胁迫也可不同程度地增加HvFer1的表达量。     

绿豆根尖细胞微管骨架有丝分裂时相发育变化的研究

提纯猪脑微管蛋白,制备兔抗微管蛋白抗血清,以此抗体与羊抗兔ｌｇＧ－ＦＩＴＣ因清,对绿豆根尖细胞进行间接免疫荧光标记和荧光显微镜检,得到了绿豆根尖细胞有丝分裂微管骨架周期发育变化的时相,如：早前期带,纺棰体微管,成膜体微管等,结果证明了双子叶植物具有与单子叶植物相似的细胞分裂微管周期时相,表明了微管架周期时相变化在高等植物中具有普遍性和共同变化的规律,讨论了微管骨架时相发育变化与染色有丝分裂行为的关     

Uptake of amino acids and peptides by developing barley embryos

Developing embryos of barley ( Hordeum vulgare L. cv. Bomi) detached 21–27 days after anthesis took up 1 mM [¹⁴C]-glutamine at pH 5 and 30°C at a rate of about 20 nmol embryo^−l h⁻¹ (5 μol g⁻¹h⁻¹). The uptake was inhibited by about 50% by di-nitrophenol and by about 80% by 300 m M unlabelled glutamine or alanine. The bulk of the uptake appeared, therefore, to be due to carrier-mediated active transport. The pH optimum of the uptake was 4.5. Leucine, proline, lysine, arginine and as-paragine were taken up at approximately similar rates as glutamine, and they also inhibited the uptake of glutamine. This, suggests that the uptake of glutamine was at least partly due to an unspecific carrier(s) also shared by other amino acids. The embryos also took up the dipepti.de glycykarcosine; the rate was about 6 nmol embryo⁻¹h⁻¹ (1.5 μol g⁻¹h⁻¹) (2 mM glycylsarcosine, pH 4.5, 30°C). The uptake was inhibited by about 70% by dinitrophenol or by 300 m M glycylglycine. This indicates that the bulk of the uptake was due to carrier-mediated active transport. The pH optimum of the uptake was about 4.5.
The rates of glutamine and glycylsarcosine uptake increased during the early and middle stages of embryo development (until day 28 after anthesis), but decreased towards the end of the maturation of the grain. These changes, as well as the relatively high activities, suggest that carrier-mediated active uptake of amino acids, and possibly also that of peptides, plays a role in the nutrition of the developing embryo.     

Temperature-induced absorbance changes in developing barley chloroplasts

Absorbance changes on cooling and heating of barley ( Hordeum vulgare L. cv. IB65) chloroplasts greened for 12, 48 and 72 h were investigated to understand the structural changes during biogenesis of chloroplast membranes. Upon cooling the chloroplast suspension from 24 to 8°C, a positive absorbance change occurred at 678, 435 and 495 nm in 12, 48 and 72 h greened chloroplasts. During heating from 24 to 45°C negative absorbance changes were observed with some shifts in positions in different chloroplast preparations and a simultaneous increase in absorbance between 690 and 735 nm. For chloroplasts developed for 12, 48 and 72 h the changes in absorbance on cooling were 3.8, 3.3 and 1.9% at 678 nm, and on heating, 8.9, 8.3 and 4.1% at 680 nm.
The differences in absorbance changes are considered as an indication of variations in the structural organization and composition of developing chloroplasts. The reversibility of the absorbance changes was maximum in chloroplasts greened for 72 h and minimum in chloroplasts greened for 12 h. This would suggest that fully developed chloroplasts have more flexibility towards temperature-induced changes in the membranes.     

DNA replication and the development of preprophase bands in soybean protoplast cultures

Nuclear DNA replication and the development of preprophase bands (PPBs) are two chronologically close processes during the higher plant cell cycle. However, it is not clear whether occurrence of PPBs is coupled with DNA replication. A soybean protoplast culture with a high frequency of PPBs was used to study the relationship between the two processes when treated with aphidicolin, a potent and specific inhibitor of eukaryotic DNA polymerase-α. When DNA replication was partially inhibited by 10 mg l^-1 aphidicolin, both the percentage of cells with PPBs and the mitotic index (MI) decreased in absolute terms, but there were proportionately more PPBs than mitoses. Since PPBs change in appearance as they develop, they were divided into categories of early (interphase associated) and late (prophase associated). The increased PPB/MI ratio was associated with an increased proportion of early stage PPBs relative to late stage PPBs. When DNA replication was completely blocked by 50 mg l^-1 aphidicolin, both MI and the percentage of cells with PPBs were close to zero. These results suggest that development of PPBs was to a large extent coupled DNA replication. We propose that the increased PPB/MI ratio at 10 mg l^-1 aphidicolin was due to a linkage between the duration of interphase and the time period in which early stage PPBs are visible. The increased duration of early PPBs partially compensates for the reduced number of nuclei reaching the stage of PPB initiation. Furthermore, in cultures containing aphidicolin, the percentage of PPBs with simultaneous perinuclear fluorescence (PNF, accumulation of microtubules on nuclear envelope) was reduced and whenever PNF was prominent and dense on the nuclear envelope the nucleus showed chromatin condensation. These observations indicated that the transition from PPB to PNF and then to the prophase spindle is closely related to the progress of the nuclear cycle.     

A minimal model of light-induced circadian rhythms of nitrate reductase activity in leaves of barley

Observed circadian rhythms of nitrate reductase (NR) (EC 1.6.6.1) activity in leaves of barley ( Hordeum vulgare L. cv. Herta) under continuous light conditions are described by a simple kinetic model. The oscillatory mechanism has been decomposed into the negative and positive feedback loops which are necessary according to present theories of chemical oscillating systems. Our results indicate that the decrease of NR activity in darkness can be considered as a reversible unimolecular conversion of the active form of NR into an inactive form, forming a negative stabilizing feedback loop. The light-induced increase of NR activity is related to a positive destabilizing feedback loop. In our treatment this process is represented as an autocatalytical reaction.     

Diurnal variations of nitrate reductase activity and stability in barley leaves

Diurnal variations of nitrate reductase (NR) activity and stability have been studied in leaves of barley seedlings ( Hordeum vulgare L. cv. Herta) grown in an 8 h light/16 h darkness regime. Stability (decay) of NR was tested both in the extracts and in the plants. In the morning, when the plants were transferred to light, NR activity increased rapidly during the first hour and then remained constant. After the photoperiod, activity decreased rapidly during the first hour of darkness and then remained fairly constant during the rest of the dark period. The high NR activity during the photoperiod was associated with low NR stability both in the extracts and in the plants. On the other hand the low NR activity during the dark period was associated with high stability in the extracts and in the plants.     

Factors affecting dry weight accumulation in developing barley endosperm

Some factors that may be concerned in determining final grain weight in barley ( Hordeum vulgare L. var. distichum ) have been investigated. Variation in endosperm fresh and dry weight, volume and starch content have been recorded at different stages of grain development between anthesis and harvest-ripeness for two barleys, cvs Kym and Golden Promise, differing in final grain weight. Results were recorded under both field and glasshouse conditions. The results suggest that the higher final dry weight of Kym, in comparison with Golden Promise, is a function of both rate and duration of grain filling. Only at later stages of endosperm development did the differences in volume become significant and the Kym endosperms continued to increased rapidly in volume for two to three days after endosperm volume had reached a maximum in Golden Promise. The rates of starch accumulation in both cultivars were very similar but starch deposition continued in Kym endosperms for four to five days after deposition in Golden Promise endosperms had slowed down.