Origin of the quiescent center in the root of Capsella bursa-pastoris (L.) Medik

The origin of the quiescent center in the embryonic radicle of Capsella bursa-pastoris was investigated by in-situ hybridization to cellular polyadenylic-acid-containing RNA using [³H]polyuridylic acid as a probe. In the globular embryo, autoradiographic silver grains were localized in all cells of the presumptive root apex except in the hypophysis. As the inner cell formed by a transverse division of the hypophysis cut off new cells toward the central procambial cylinder of the embryo, these cells remained characteristically unlabeled, in contrast to the labeled cells of the rest of the embryo. In the embryonic radicles of mature seeds and of seedlings, cells derived from the hypophysis appeared as a nonmeristematic, unlabeled, hemispherical group, bounded by the procambium to the inside and the root epidermis to the outside. When root tips excised from 2-d-old seedlings were incubated in [methyl-³H]thymidine, sectioned, and autoradiographed, cells derived from the inner cell of the hypophysis were found to be unlabeled, thus showing that they constitute the specific cells of the quiescent center. These results present evidence for the single-cell origin of the quiescent center in an angiosperm root and a role for the hypophysis in it.Abbreviations poly(A)⁺RNA polyadenylicacid-containing RNA - [³H]poly(U) [³H]polyuridylic acid - QC quiescent center This work was supported in part by National Science Foundation grants PCM-7902898 and DCB-8709092.     

NAD-dependent aromatic alcohol dehydrogenase in wheats (Triticum L.) and goatgrasses (Aegilops L.): evolutionary genetics

Summary Evolutionary electrophoretic variation of a NAD-specific aromatic alcohol dehydrogenase, AADH-E, in wheat and goatgrass species is described and discussed in comparison with a NAD-specific alcohol dehydrogenase (ADH-A) and a NADP-dependent AADH-B studied previously. Cultivated tetraploid emmer wheats (T. turgidum s. l.) and hexaploid bread wheats (T. aestivum s. l.) are all fixed for a heterozygous triplet, E^0.58/E^0.64. The slowest isoenzyme, E^0.58, is controlled by a homoeoallelic gene on the chromosome arm 6AL of T. aestivum cv. Chinese Spring and is inherent in all diploid wheats, T. monococcum s. Str., T. boeoticum s. l. and T. urartu. The fastest isoenzyme, E^0.64, is presumably controlled by the B- and D-genome homoeoalleles of the bread wheat and is the commonest alloenzyme of diploid goat-grasses, including Ae. speltaides and Ae. tauschii. The tetraploid T. timopheevii s. str. has a particular heterozygous triplet E^0.56/E^0.71, whereas the hexaploid T. zhukovskyi exhibited polymorphism with electromorphs characteristic of T. timopheevii and T. monococcum. Wild tetraploid wheats, T. dicoccoides and T. araraticum, showed partially homologous intraspecific variation of AADH-E with heterozygous triplets E^0.58/E^0.64 (the commonest), E^0.58/E^0.71, E^0.45/E^0.58, E^0.48/E^0.58 and E^0.56/E^0.58 recorded. Polyploid goatgrasses of the D-genome group, excepting Ae. cylindrica, are fixed for the common triplet E^0.58/E^0.64. Ae. cylindrica and polyploid goatgrasses of the C^u-genome group, excepting Ae. kotschyi, are homozygous for E^0.64. Ae. kotschyi is exceptional, showing fixed heterozygosity for both AADH-E and ADH-A with unique triplets E^0.56/E^0.64 and A^0.49/A^0.56.     

Glutamate dehydrogenase from Pisum sativum L.

A 2–8-fold increase in the activity of glutamate dehydrogenase (GDH), accompanied by an alteration of the GDH isoenzyme pattern, was observed in detached pea shoots floated on tap water (preincubated shoots). Sugars supressed the process, whereas NH ₊ ⁴ and various metabolites as well as inhibitors of energy metabolism and protein synthesis were ineffective. The subcellular distribution pattern revealed evidence that the GDH isoenzymes are exclusively located in the mitochondrial matrix. The alterations in GDH activity occurring in preincubated shoots are restricted to the mitochondria.An experimental device suitable for studying the GDH function in isolated intact mitochondria has been established. Using [¹⁴C] citrate as the carbon source and hydrogen donor, the mitochondria synthesized considerable amounts of glutamate upon addition of NH ₊ ⁴ . The rates of glutamate formation in dependency of increasing NH ₊ ⁴ levels follow simple Michaelis-Menten kinetics. Half-saturation concentrations of NH ₊ ⁴ of 3.6±1.2 mM; 1.9±0.06 mM and 1.6±0.1 mM were calculated for the mitochondria isolated from pea shoots, roots, and preincubated shoots, respectively. The results are discussed in relation to the possible role of GDH in NH_+/4 assimilation at elevated intracellular NH_+/4 levels.Abbreviations GDH Glutamate dehydrogenase - MDH malate dehydrogenase - GOT aspartate aminotransferase - SDH succinate dehydrogenase - HEPES 4-(2-hydroxyethyl)-1-piperazineethan-sulfonic acid - BSA bovine serum albumin - TPP thiamine pyrophosphate - DNP 2,4-dinitrophenol - CCCP carbonyl cyanide m-chlorophenylhydrazone - DCPIP 2,6-dichlorophenolindophenol Dedicated to Professor Dr. Maximilian Steiner on the occasion of his 75th birthday     

Localisation and characterisation of homoserine dehydrogenase isolated from barley and pea leaves

Two forms of homoserine dehydrogenase exist in the leaves of both barley and pea; one has a large molecular weight and is inhibited by threonine, the other is of smaller molecular weight and insensitive to threonine but inhibited by cysteine. The subcellular localisation of these enzymes has been examined. Both plants have 60–65% of the total homoserine dehydrogenase activity present in the chloroplast and this activity is inhibited by threonine. The low molecular weight, threonine-insensitive form is present in the cytoplasm. Total homoserine dehydrogenase activity from barley leaves showed progressive desensitisation towards threonine with age in a similar manner to that previously described for maize. It was shown that the effect was due to desensitisation of the chloroplast enzyme, and not to an increase in the insensitive cytoplasm enzyme. No corresponding desensitisation to threonine was detected in pea leaves. The different forms of homoserine dehydrogenase could be separated from pea leaves by chromatography on Blue Sepharose; the threonine-sensitive enzyme passed straight through and the threonine insensitive form was bound. A similar separation of the barley leaf isoenzymes was obtained using Matrex Gel Red A affinity columns; in this case however, the threonine-sensitive isoenzyme was bound. In both plants, the threonine insensitive isoenzyme was subject to greater inhibition by cysteine than was the threonine-sensitive isoenzyme.Abbreviation HSDH homoserine dehydrogenase     

Light-stimulated cell expansion in bean (Phaseolus vulgaris L.) leaves

Cell expansion in dicotyledonous leaves is strongly stimulated by bright white light (WL), at least in part as a result of light-induced acidification of the cell walls. It has been proposed that photosynthetic reactions are required for light-stimulated transport processes across plasma membranes of leaf cells, including proton excretion. The involvement of photosynthesis in growth and wall acidification of primary leaves of bean has been tested by inhibiting photosynthesis in two ways: by reducing chlorophyll content of intact plants with tentoxin (TX) and by treating leaf discs with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Exposure to bright WL stimulated growth of intact leaves of TX-treated plants. Discs excised from green as well as from TX-or DCMU-treated leaves also responded by growing faster in WL, as long as exogenous sucrose was supplied to the photosynthetically inhibited tissues. The WL caused acidification of the epidermal surface of intact TX-leaves, but acidification of the incubation medium by mesophyll cells only occurred when photosynthesis was not inhibited. It is concluded that light-stimulated cell enlargement of bean leaves, and the necessary acidification of epidermal cell walls, are mediated by a pigment other than chlorophyll. Light-induced proton excretion by mesophyll cells, on the other hand, may require both a photosynthetic product (or exogenous sugars) and a non-photosynthetic light effect.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1 -dimethylurea - OC osmotic concentration - RL red light - TX tentoxin - WL white light We thank Dr. G.E. Templeton, University of Arkansas, Fayetteville, USA, for initially supplying us with TX, and also Dr. Stephen O. Duke, Southern Weend Science Laboratory, Stoneville, Miss., USA, for suggesting this compound for our experiments. We are grateful to Professor E. Ballio for his generous gift of fusicoccin.     

Somatic embryogenesis from cell suspension and protoplast cultures ofCapsella bursa-pastoris (L.) Medic

Summary Rapidly growing cell suspension cultures of shepherd’s purse (Capsella bursa-pastoris L. Medic.) were established from leaf-derived calli. These suspensions remained unorganized in the presence of 2,4-D, but underwent extensive root organogenesis in a growth regulator-free liquid medium. Attempts to induce direct embryogenesis in liquid cultures were unsuccessful, but numerous embryos were obtained from cells plated onto growth-regulator-free solid medium. These embryos were frequently abnormal, and secondary embryogenesis was problematic for plant recovery but fertile plants were recovered. Viable protoplasts could readily be isolated from these cell suspensions. After 1 wk of culture, protoplast viability was 62%, and 7% of the cells had divided. Embryogenesis was observed from protoplast-derived microcolonies, plated on growth-regulator-free medium. Although these somatic embryos were difficult to root, plants were recovered. New cell suspensions were more recently established, which were only 4 to 6 mo. old when plant regeneration was attempted. Numerous shoots were obtained when these cells were plated onto growth-regulator-free solid media. However, these shoots differed from the embryos previously obtained in that they readily rooted and rapidly developed into plantlets. This system may allow the use of shepherd’s purse as a gene source for introgression of agronomically interesting traits intoBrassica crop species through protoplast manipulation and somatic hybridization.     

Evolution of the tetraploidCapsella bursa-pastoris (Brassicaceae): Isoelectric focusing analysis of Rubisco

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) and its subunits (large subunits = LSU, small subunits = SSU) were isolated from threeCapsella spp. by gel electrophoresis and polypeptide composition was analyzed by isoelectric focusing (IEF) in the presence of 8M urea. The described techniques are recommended for large scale systematic studies. Multiple IEF banding patterns of the SSU are probably the outcome of a heterogenous multigene family. The two diploid speciesC. rubella andC. grandiflora show an identical IEF pattern and could be differentiated from the putative allotetraploidC. bursa-pastoris only by the SSU banding pattern. Uniqueness of some SSU bands in the tetraploid and in the two diploid species, respectively, may indicate an ancient alloploid origin of tetraploidC. bursa-pastoris followed by events leading to divergences in the genomes of the allotetraploid and its presumed diploid progenitors after the hybridization event (SSU gene elimination, acquisition of new SSU genes).     

Synchronization of protoplasts from Glycine max (L.) Merr. and Brassica napus (L.)

Cells of Glycine max originating in a suspension culture and cells of Brassica napus prepared from hypocotyls were synchronized. Synchronization was achieved by preparing protoplasts in the usual way and subsequently letting the protoplasts regenerate into cells by removing the cell-wall-digesting enzymes. More than 70% of the cells had divided synchronously at the end of the first cycle as determined by the mitotic index. The high frequency of mitosis critically depended on the osmolality of the medium. The duration of the S-phase was estimated by measuring the activity of thymidylate kinase as well as incorporation of [³H]deoxythymidine into acid-insoluble material. The data indicate that synchronization is induced by resetting the cell cycle.Abbreviations dTMP deoxythymidine 5-monophosphate - TCA trichloroacetic acid     

Production and genetic analysis of partial hybrids in intertribal crosses between Brassica species (B. rapa, B. napus) and Capsella bursa-pastoris

Capsella bursa-pastoris (L.) Medic (2n = 4x = 32) is a natural double-low (erucic acid < 1%, glucosinolates < 30 micromol/g) germplasm and shows high degree of resistance to Sclerotinia sclerotiorum. Hybridizations were carried out between two Brassica species viz. B. rapa (2n = 20) and B. napus (2n = 38) as female and C. bursa-pastoris as male parent to introduce these desirable traits into cultivated Brassica species. Majority of F(1) plants resembled female parents in morphology and only a few expressed some characters of male parent, including the white petals. Based on cytological observation of somatic cells, the F(1) plants were classified into five types: two types from the cross with B. rapa, type I had 2n = 27-29; type II had 2n = 20; three types from the crosses with B. napus, type III was haploids with 2n = 19; type IV had 2n = 29; type V had 2n = 38. One to two chromosomes of C. bursa-pastoris were detected in pollen mother cells (PMCs) of type I plant by genomic in situ hybridization (GISH), together with chromosomal segments in ovary cells and PMCs of some F1 plants. Amplified fragment length polymorphism (AFLP) bands specific for the male parent, novel for two parents and absent bands in Brassica parents were generated in majority of F1 plants, even in Brassica-types and haploids, indicating the introgressions at various levels from C. bursa-pastoris and genomic alterations following hybridization. Some Brassica-type progeny plants had reduced contents of erucic acid and glucosinolates associated with improved resistance to S. sclerotiorum. The cytological and molecular mechanisms behind these results are discussed.     

A growth-inhibitory substance exuded from freeze-dried mesquite (Prosopis juliflora (Sw.) DC.) leaves

When lettuce seeds were incubated in a concentric circle aroundfreeze-dried mesquite leaves in a dish containing agar culture medium, thegrowth, especially radicle growth, of the seedlings was inhibited, the morestrongly the closer they were to the leaves. This result indicates thatallelopathic substance(s) inhibiting the lettuce growth are exuded from themesquite leaves. A potent substance was isolated from the exudates of thefreeze-dried mesquite leaves and identified as L-tryptophan by spectralanalyses. L-Tryptophan inhibited the radicle growth of lettuce and barnyardgrass at concentrations greater than1.5×10^–3 . The content ofL-tryptophan in the exudates of freeze-dried mesquite leaves (1 eq.) was 4.8×10^–3 . Theseresults suggest that L-tryptophan may play an important role in the allelopathyof mesquite leaves.     

The induction of sun and shade leaves of the European beech (Fagus sylvatica L.): anatomical studies

Summary Primordia from buds of sun and shade twigs of European beech (Fagus sylvatica L.) were collected six times a year for anatomical investigations. Differentiation into sun-leaf and shade-leaf primordia was first observed in early August. Sun-leaf primordia had five, and shade-leaf primordia four layers of mesophyll meristem cells. With potted graft unions of beeches possible structural changes of leaf primordia were investigated. Trees adapted to shade develop sun-leaf primordia when put into full daylight, provided the transfer happened before July. Trees adapted to full daylight developed leaf primordia which remained structurally sun-leaf primordia when the plant was kept under shade conditions. Shadeleaf branches of young beech trees cut in February in order to expose the shade buds to full daylight developed either shade leaves or intermediate shade/sun leaves. These experiments show that the subtending leaf may provide the developing axillary bud with photoassimilates, but its character, whether sun or shade leaf, has no influence on the character of the developing leaf primordia.     

In vitro plant regeneration from leaves and internode sections of sweet cherry cultivars (Prunus avium L.)

Regeneration of adventitious shoots from leaves and, for the first time, from internode sections were compared and optimized for five economically important sweet cherry cultivars, i.e. Schneiders, Sweetheart, Starking Hardy Giant, Kordia and Regina (Prunus avium L.). The influence of basal media, carbon source, combination and dosage of phytohormones, ethylene inhibitor such as silver thiosulfate and a 16 h:8 h light:dark photoperiod versus complete darkness were evaluated. Both, DKW/WPM (1:1) and Quoirin/Lepoivre (QL) basal media stimulated organogenesis more than QL/WPM (1:1), Chee and Pool (CP), Murashige Skoog (MS), Driver and Kuniyuki (DKW) or woody plant (WPM) media did. An induction phase in darkness resulted in lower or zero regeneration rates. The best regeneration efficiencies were generally obtained with thidiazuron in combination with indole-3-butyric-acid. The addition of silver thiosulfate resulted in a similar or reduced regeneration efficiency. Significant genotypic variability in adventitious bud formation was evident for both explant sources, leaf and internode section. Adventitious shoots were obtained from 11% of leaf explants and 50% of internode sections indicating that shoot regeneration from internodes was significantly more efficient than from leaves.     

Spatial distribution of mRNA during pre-fertilization ovule development in Capsella bursa-pastoris

Summary In situ hybridization of sections of the ovary and ovule of Capsella bursa-pastoris with ³H-polyuridylic acid [³H-poly(U)] showed the presence of polyadenylic acid-containing RNA [poly(A) + RNA] in the cells of the placenta and nucellus. During megasporogenesis there was a decrease in ³H-poly(U) binding activity of the nucellar cells concomitant with the appearance of poly(A) + RNA in the integuments. As the typical eight-nucleate embryo sac was formed, ³H-poly(U) binding was not apparent around the quartet of nuclei at the chalazal end, while it persisted at the micropylar end. Both the egg and synergids as well as the chalazal proliferating tissue showed high concentrations of poly(A) + RNA in their cytoplasm. The results suggest a role for transient localizations of poly(A) + RNA during female sporogenesis and gametogenesis in C. bursa-pastoris.     

定点饱和突变提高Lactobacillus casei L-乳酸脱氢酶对苯丙酮酸的催化效率

【背景】光学纯L-苯乳酸是一种天然防腐剂,也是一种高附加值的手性分子,在食品、制药和材料等领域有广阔的应用前景。本实验室已发现来源于Lactobacillus casei CICIM B1192的NADH依赖型L-乳酸脱氢酶(L-LcLDH)可不对称还原苯丙酮酸制备L-苯乳酸,但其活性较低。为提高L-LcLDH催化苯丙酮酸的催化效率,构建了一个单突变体L-LcLDH~(Q88R),其催化效率kcat/Km是L-LcLDH的4.9倍。【目的】为进一步提高L-LcLDH~(Q88R)催化苯丙酮酸的催化效率,采用饱和突变技术将位于L-LcLDH~(Q88R)底物结合口袋附近的氨基酸残基Ile~(229)随机替换为其他氨基酸,以获得活性更高的优良突变体。【方法】以重组表达质粒p ET-22b-LcldhQ88R为模板,采用全质粒PCR技术对L-LcLDH~(Q88R)基因(LcldhQ88R)中编码Ile~(229)的密码子实施饱和突变,构建突变转化子文库。以催化苯丙酮酸的活性为指标,从文库中筛选出优良的突变转化子。【结果】突变转化子(Escherichia coli/Lcldh~(Q88R/I229Q))表达出一种由Arg和Gln分别替换了Gln88和Ile~(229)的双突变体L-LcLDH~(Q88R/I229Q)。重组表达产物L-LcLDH~(Q88R/I229Q)的酶学性质分析表明:L-LcLDH~(Q88R/I229Q)的比活性是L-LcLDH的18.5倍,是L-LcLDH~(Q88R)的2.3倍;其催化效率分别为后两者的6.8倍和1.4倍。L-LcLDH突变前后的温度和pH特性改变不大。根据分子对接结果推测出,双突变Q88R/I229Q导致L-LcLDH的底物结合口袋的入口变大和构型的变化可能对其催化活性的提高发挥了重要作用。【结论】双突变Q88R/I229Q显著提高了L-LcLDH的活性和催化效率,使得L-LcLDH~(Q88R/I229Q)在不对称还原苯丙酮酸制备L-苯乳酸中成为有潜力的工具酶。     

The control of respiration in wheat (Triticum aestivum L.) leaves

The aim of this work was to discover whether the respiration of wheat (Triticum aestivum L. cv. Huntsman) leaves, transferred to darkness after 7 h photosynthesis, showed an initial period of wasteful respiration. For young and old leaves, CO₂ production and O₂ uptake after 7 h photosynthesis were up to 56% higher than at the end of an 8-h night. The maximum catalytic activities of citrate synthase (EC 4.1.3.7), aconitase (EC 4.2.1.3), fumarase (EC 4.2.1.2) and cytochrome-c oxidase (EC 1.9.3.1) at the end of the day did not differ from those at the end of the night. Changes in the contents of glucose 6-phosphate, fructose-1,6-bisphosphate, dihydroxyacetone phosphate, and -ketoglutarate did not as a group parallel the changes in the rate of respiration. The detailed distribution of label from [U-¹⁴C] sucrose supplied to leaves in the dark was similar at the end of the day and the end of the night. No correlation was observed between the rates of leaf respiration and extension growth. It is argued that the higher rate of respiration at the beginning of the night cannot be attributed to wasteful respiration.Abbreviation RQ respiratory quotient We thank Dr H. Thomas and Professor C.J. Pollock, Institute for Grassland and Environmental Research, Plas Gogerddan, Aberystwyth, UK for their generous help in measuring leaf extension. R.H.A. thanks the Science and Engineering Research Council for a studentship.     

Water use by the desert cucurbit Citrullus colocynthis (L.) Schrad.

Summary The rates of water use and leaf surface conductance of Citrullus colocynthis (Cucurbitacea) were evaluated from measurements of the surface temperature and microenvironment of leaves. At desert sites in Saudi Arabia the transpiration rates reached 0.13–0.17 g m^-2 s^-1 and the leaf temperatures were always close to air temperature. Leaf models (dry) placed in the canopy were considerably warmer than the air. To investigate responses over a wider range of conditions, plants were grown in a controlled environment room. It was found that when conditions were made hotter than those that occurred in the desert, the stomatal conductance increased greatly. Transpiration rate attained 0.6 g m^-2 s^-1 and the leaves were up to seven degrees cooler than the air. The results suggest a finely-tuned control mechanism working like a switch when the leaves experience extreme conditions, and enabling the plant to avoid lethal temperatures.     

Plant regeneration from cotyledonary protoplasts of cauliflower (Brassica oleracea L. var.botrytis L.)

Summary Protoplasts isolated enzymatically from precultured cotyledonary leaves ofB. oleracea var.botrytis and cultured in KM8p medium (Kao andMichayluk 1975) underwent sustained divisions in about 0.1% population to eventually produce callus, whereas mesophyll protoplasts from either field grown orin vitro raised plants failed to divide. The callus readily differentiated on Murashige-Skoog medium as modified for shoot culture (Binding 1974) to give rise to shoot and roots.