Uptake of glutamine by the scutellum of germinating barley grain

Scutella separated from germinating grains of barley (Hordeum vulgare L. cv Himalaya) took up [¹⁴C]glutamine at an initial rate of about 10 micromoles·gram⁻¹·hour⁻¹ in the standard assay conditions (pH 5, 30°C, 1 millimolar glutamine). Inhibition by unlabeled glutamine and by dinitrophenol indicated that about 95% of the uptake was due to carrier-mediated active transport. The pH optimum of the uptake was 5, and after correction for a nonmediated component the uptake appeared to conform to Michaelis-Menten kinetics with an apparent K_m of about 2 millimolar and a V_max of about 25 micromoles·gram⁻¹·hour⁻¹.

The uptake of glutamine was inhibited by all of the 18 amino acids tested; the mode of inhibition was studied only with proline and was competitive. Eight of the ten amino acids tested at high concentrations appeared to be able to inhibit the mediated uptake of glutamine virtually completely. However, when the inhibitory effect of asparagine was extrapolated to an infinitely high concentration of asparagine, about 24% of the mediated uptake of glutamine remained uninhibited. These results suggest that glutamine is taken up by two (or more) rather unspecific amino acid uptake systems, the minor one having no affinity for asparagine.

Glutamine and alanine could completely inhibit the mediated uptake of 1 millimolar leucine, but about 12% of the mediated uptake appeared to be uninhibitable by asparagine. Furthermore, the ratio of the mediated uptake of glutamine to that of leucine changed from 0.9 to 1.7 between days 1 and 3 of germination. These results give further support for the presence of two unspecific amino acid uptake systems in barley scutella.

     

Uptake of proline by the scutellum of germinating barley grain

Scutella separated from germinating grains of barley (Hordeum vulgare L. cv Himalaya) took up 1 millimolar l-[¹⁴C]proline at an initial rate of about 6.5 micromoles gram⁻¹ fresh weight hour⁻¹ (pH 5, 30°C). The uptake had a pH optimum at 5. The bulk of the uptake (93%) was via carrier-mediated active transport. All of the 19 l-amino acids tested at 10 millimolar concentration inhibited the mediated uptake of 1 millimolar proline, the inhibitions varying from 18 to 76%. By studying how large a fraction of the mediated uptake was inhibitable by asparagine, alanine, glutamine, and leucine, the mediated uptake was shown to be due to three components. Two of these are most probably attributable to the two nonspecific uptake systems proposed earlier to act in the uptake of glutamine and leucine. The third component was not inhibited by glutamine, asparagine, or alanine, but was inhibited by unlabeled proline and leucine. The uptake by this system was apparently carrier-mediated active transport. d-Proline inhibited this system as strongly as l-proline. Nine of the 16 l-amino acids tested at 50 millimolar concentrations did not inhibit the uptake of 1 millimolar proline by this system. Valine, leucine, isoleucine, and the basic amino acids were inhibitory, but in spite of this, they did not appear to be taken up by this system. It seems therefore that in addition to two nonspecific amino acid uptake systems the scutella have an uptake system which is specific for proline. It is likely that this proline-specific system accounts for the bulk of proline uptake in a germinating grain.     

Uptake of glycylglycine by the scutellum of germinating barley grain

The scutella separated from germinating barley grains (Hordeum vulgare L. cv. Himalaya) took up the dipeptide [¹⁴C]glycylglycine (Gly-Gly) rapidly from incubation media. The pH optimum of the process was about 4.5, and the rate of uptake conformed to Michaelis-Menten kinetics with an apparent K_m of 2.3 mm and V_max of 41 μmole gram⁻¹ hour⁻¹. The uptake was strongly inhibited by dinitrophenol and cyanide and by lack of O₂.     

Characteristics and development of leucine transport activity in the scutellum of germinating barley grain

Scutella separated from grains of Himalaya barley after germination for 3 days rapidly took up l-leucine from aerated incubation media; with 1 millimolar leucine the rate varied between 4 and 14 micromoles per gram per hour and the pH optimum was at 3.5 to 5, both depending on buffer composition and prewashing time. The rate of the uptake increased with increasing concentration of leucine in a complex manner, which could be interpreted as multiphasic kinetics with apparent K(m) values of 3.4 and 15.5 millimolar below and above 3 millimolar leucine, respectively. The uptake took place against a concentration difference (highest estimated ratio 270: 1) and was strongly inhibited by dinitrophenol. Uptake was apparently due to active transport requiring metabolic energy.The development of the uptake activity during germination was studied using Pirkka barley. A low activity was present in the scutella of ungerminated grains. It began to increase after 6 hours imbibition, and the increase was biphasic, the major changes occurring during days 0 to 3 and 4 to 6. The total increase was about 20-fold.The regulation of the development was studied by allowing separated embryos to germinate on agar gel. The increase of uptake activity was strongly inhibited by inhibitors of RNA or protein synthesis. Increase did not require the presence of the embryo proper, and was not affected by gibberellic or abscisic acid. Removal of the endosperm greatly accelerated the increase of uptake activity, and the presence of 5 or 20 millimolar glutamine counteracted the removal of the endosperm. The results suggest that the availability of glutamine or amino acids in general in the endosperm may regulate the development or the activity of the transport system.     

Secretion of alpha-amylase by the aleurone layer and the scutellum of germinating barley grain

α-Amylase activities in extracts of different parts of barley grain (Hordeum vulgare L. cv Himalaya) were low after 1 day of germination at 20°C, but they began to increase afterwards. In the scutellum and the aleurone layer, the increases were small, but in the starchy endosperm a great increase took place between days 1 and 6.

When the aleurone layers were separated from germinating whole grains and incubated in 10 millimolar CaCl₂, the α-amylase activity in the medium increased linearly for about 30 to 60 minutes, indicating secretion. The activity inside the aleurone layer decreased only slightly during the incubation, indicating that secretion of α-amylase was accompanied by synthesis. The rates of secretion in vitro by the aleurone layers separated at different stages of germination corresponded rather well to the rate of accumulation of α-amylase activity in the starchy endosperm in a whole grain.

Scutella separated after 1 day of germination released small amounts of α-amylase activity into 10 millimolar CaCl₂. This release was linear for at least 1 hour and did not occur at 0°C; it is therefore likely to be due to secretion. At later stages of germination, the secretion by the scutella was slower than at day 1 and the total secretion accounted for only 5 to 10% of the increase of α-amylase activity in the starchy endosperm in a whole grain.

Since the times from the separation of the parts of the grain to the beginning of the secretion assay (10-40 minutes) as well as the duration of the assay itself (20-60 minutes) were short, the rates of secretion by the separated grain parts are likely to represent those in an intact grain. The results indicate therefore that at least in the conditions used the bulk of the total α-amylase in the starchy endosperm is secreted by the aleurone layer, the contribution by the scutellum being only 5 to 10% of the total activity.

     

Characteristics of the protein carrier of the peptide-transport system in the scutellum of germinating barley embryos

Through the use of the protein reagents N-ethylmaleimide, p-chloromercuribenzenesulphonic acid and phenylarsine oxide, it is shown that in the scutellum of the germinating barley embryo, the transport of peptides, but not the transport of amino acids or glucose is specifically thiol-dependent. Furthermore, these essential thiol groups are shown to exist as redox-sensitive, vicinal-dithiols that lie at the substrate-binding sites of the peptide-transport proteins. The binding of N-ethylmaleimide to these dithiols is shown to be very fast, matching the kinetics of inhibition of peptide transport by this reagent. A technique for the specific labelling of the dithiols with N-ethyl[2,3-¹⁴C]maleimide is described, which allows the carrier proteins to be visualized at the scutellar epithelium using radioautography and permits calculation of the approximate amount of peptide-transport protein present per scutellum. In related studies, the importance of arginyl and histidyl residues to both amino-acid and peptide transport is shown, although other residues, e.g. carboxyl ligands do not seem to be critically involved.Abbreviations Ala alanine - Gly glycine - Leu Leucine - NEM N-ethylmaleimide - PAO phenylarsine oxide - PCMBS p-chloromercuribenzenesulphonic acid - Phe phenylalanine     

Regulation of development of leucine uptake activity by glutamine in the scutellum of germinating barley grain

Scutella from ungerminated grains of barley (Hordeum vulgare L. cv Pirkka) take up leucine at a slow rate, which increases rapidly during germination. When endosperms were removed from the grains after imbibition for 4 hours or after germination for 12 or 72 hours, the increase in the rate of leucine uptake was greatly accelerated during subsequent incubation of the embryos or scutella. These increases were rapidly inhibited by cordycepin and cycloheximide, suggesting that protein synthesis, probably synthesis of the carrier protein, was required for the development of the uptake activity.

In separated embryos or scutella, the increases in the leucine uptake activity were inhibited by glutamine. The inhibitions caused by glutamine and cycloheximide were not additive, suggesting that glutamine did not interfere with the function of the carrier but repressed its synthesis. Glutamine did not inhibit the simultaneous increase in peptide uptake; in this respect, its effect was specific for leucine uptake, which appears to be due to a general amino acid uptake system.

Some other protein amino acids also inhibited the increase in leucine uptake without inhibiting the increase in peptide uptake. However, these effects were smaller than that of glutamine.

These results suggest that the transfer of leucine (and other amino acids) from the endosperm to the seedling in a germinating barley grain is regulated at the uptake step by repression of the synthesis of the amino acid carrier protein by glutamine and—possibly to a lesser extent—by some other amino acids taken up from the endosperm.

     

Taxonomic differences among closely related pines Pinus sylvestris, P. mugo, P. uncinata, P. rotundata and P. uliginosa as revealed in needle sclerenchyma cells

The purpose of the present study was to test the taxonomic value of sclerenchyma in distinguishing Pinus sylvestris and P. mugo, P. uncinata, P. rotundata and P. uliginosa, all representing the subsection Sylvestres within the genus Pinus. Thirty-six samples were gathered in natural populations. Every sample was represented with 30 individuals, every individual with 10 brachyblasts. Three types of sclerenchymatic cells surrounding the resin canals and four between vascular bundles were distinguished. Relations among samples and taxa were verified using discriminant analysis and clustering based on Euclidean distances. The types of sclerenchymatic cells surrounding the resin canals and located between the vascular bundles differentiate the compared taxa when used as average frequencies but are extremely variable and do not allow the classification of every individual. The study demonstrated that the type of sclerenchymatic cells surrounding the resin canals and between the vascular bundles in needles could have an important taxonomic value in distinguishing the taxa of two-needle pines of the subsection Sylvestres in Europe at the population level. The distinguishing of individuals was difficult because of very high variation of sclerenchyma characters.     

Isolation and identification of proteins from the peptide-transport carrier in the scutellum of germinating barley (Hordeum vulgare L.) embryos

Peptide-transport proteins, intrinsic to the epithelial plasmalemmae of the scutella of germinating barley (Hordeum vulgare L.) embryos, have been selectively labelled with p-chloro-[²⁰³Hg]mercuribenzenesulphonate using both a substrate-screening technique and a procedure developed to label exclusively vicinal dithiol groups, which were shown previously (Walker-Smith and Payne, 1983, FEBS Lett. 160, 25–30) to be essential components of the peptide-transport system. After radioactive labelling, proteins from the scutellar membranes have been solubilised with lithium diiodosalicylate plus sodium dodecyl sulphate and separated by using polyacrylamide gel electrophoresis. Fluorography and silver staining of these gels has for the first time allowed identification of two presumptive components of the peptide-transport system. These components only become detectable in an extract of the scutellar epithelia after 15 h imbibition, concomitant with a dramatic increase in peptide-transport activity, and they remain present at least 3 d after the onset of germination. [³⁵] Methionine was shown to be incorporated into these proteins between 15–20 h after imbibition, but its incorporation during a similar 5 h period into scutella isolated after 3 d was undetectable, implying a slow turnover of these proteins during the later stages of germination.Abbreviations Ala₂, Ala₃ dialanine, trialanine - CHAPS 3-((3-cholamidopropyl) dimethylammonio)-1-propanesulphonate - p-CMBS p-chloromercuribenzenesulphonic acid - NEM N-ethylmaleimide - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

trxS基因对大麦发芽籽粒中蛋白质降解的影响

对转trxS基因大麦籽粒发芽过程中蛋白酶活性、不同蛋白组分含量和贮藏蛋白SDS-PAGE图谱的变化进行了研究。结果表明：与对照相比,转基因籽粒中的蛋白酶活性提高;清蛋白、球蛋白、醇溶蛋白和谷蛋白含量低于对照。SDS-PAGE图谱也表明,转基因籽粒中贮藏蛋白降解快于对照。     

湿地松×洪都拉斯加勒比松遗传多样性的ISSR分析

杂交育种是产生遗传变异、表型变异及选择新变异的重要方法。然而系统发育不清晰,选择较近的亲缘关系亲本用于杂交子代往往表现出较低的遗传多样性。为探究湿地松、洪都拉斯加勒比松种间杂交后代遗传多样性水平,对8个湿地松×洪都拉斯加勒比松家系进行ISSR分析。利用10条引物共产生60个表达清晰可用于分析的标记,其中48个标记表现为多态性,占总标记数的80%; 湿加松各个家系多态位点百分率在5%～23.33%之间; 各个家系基因多样性指数在 0.015 2～0.087 2之间,Shannon指数的范围在 0.021 6～0.129 4之间(家系水平为 0.293 4)。8个家系间的基因分化系数G_st为0.743 5,即总的遗传变异中有74.35%的变异存在于家系间,家系内的遗传变异占总遗传变异的25.65%。采用UPGMA法对湿加松的8个家系进行了聚类分析,确定了各个家系之间的遗传亲缘关系。8个家系间的基因流N_m为 0.172 5,表明基因流处于较低水平。     

中甸灯台报春与鹅黄灯台报春的系统演化

中甸灯台报春和鹅黄灯台报春在形态上较为相似,通常被认为是亲缘关系较近的物种。该研究选取核基因ITS序列和叶绿体trn L-F为分子标记,利用贝叶斯的方法构建系统树,在用两种分子标记构建的系统树中,两种报春均以0.81的支持率聚成一个分支,明确了两种报春的姐妹种关系。首先,通过选取两个物种的五个形态学性状,进行主成分分析,聚类结果中两种报春各自的个体分别聚在两块区域,表明两种报春的形态学性状产生了明显的分化,且中甸灯台报春的花部各性状值明显大于鹅黄灯台报春,表明花部综合征和繁育系统间的相关联系。然后,通过用8对SSR分子标记对两种报春进行STRUCTURE聚类分析,结果检测K的最适值为2,此时中甸灯台报春和鹅黄灯台报春的个体各自以不同颜色聚成界限清晰的两块区域,揭示了两种报春在分子水平上也产生了显著而稳定的分化,通过Ima2软件,依据叶绿体联合序列,计算了两种报春的分化时间大约是在更新世,推测这两个姐妹种的物种分化受更新世气候动荡和一些地质历史事件的影响。最后,运用maxent软件分析两个物种的生态位差异,结果表明最干季节降水量、年均降水量和季节性降水量对中甸灯台报春的分布有重要影响,最冷季节平均温度、最干月降水量、年均降水量和季节性降水量对鹅黄灯台报春的分布有重要影响,两个物种间的生态位产生了分化。由此推测,这种生态位的差异是物种分化的结果,同时也是物种间系统演化的重要驱动力。     

Homology of the root adhesin of Pseudomonas fluorescens OE 28.3 with porin F of P. neruginosa and P. syringae

Summary A transformation system for the thermophilic cellulolytic fungus Talaromyces sp. CL240 has been developed, using the phleomycin resistance gene from Streptoalloteichus hindustanus (Sh ble) as a dominant selectable marker. The plasmids (pAN8-1 and pUT720) carrying the Sh ble gene under the control of the Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter, allowed selection of phleomycin-resistant transformants. A new promoter sequence cloned from chromosomal DNA of Trichoderma reesei (pUT737) was also able to drive efficient expression of the Sh ble gene in Talaromyces sp. CL240, resulting in the selection of transformants that were highly resistant to phleomycin.     

应恢复赤车属头序赤车组和头序赤车

头序赤车的特征在于其具花序托和总苞的雌头状花序,根据此一特征即可将它与赤车属的其它所有种区别开。因此,在2002年被错误归并为异名的此赤车属进化种,以及根据其建立的单种进化组头序赤车组在该文中予以恢复。