Involvement of cytokinins in the germination of chick-pea seeds

Eight cytokinins detected in germinated chick-pea (Cicer arietinum L. var. Castellana) seeds were first present in the embryonic axes but appeared in the cotyledons after 12h of germination. The cytokinins detected in the cotyledons originate in the embryonic axes, but no passage of these substances from the cotyledons to the axes was detected, except when the seeds were treated with red light.It is concluded that the role played by the embryonic axis in mobilizating the main reserves of the cotyledons is mainly effected through these cytokinins. Both natural and synthetic cytokinins exert an important regulatory role in the hydrolysis of reserve proteins and calcium could be involved as an intermediate.Abbreviations BA benzyladenine - cot. cotyledon - (diH)Z dihydrozeatin - (diH)ZR dihydrozeatin riboside - GZR glycosyl zeatin riboside - 2iP 277-1 - iPA 277-2 riboside - Kin kinetin - Z zeatin - ZG zeatin glucoside - ZR zeatin riboside     

Uptake of [2-14C]abscisic acid and distribution of 14C in apple embryos

Pyrus malus L. var. Golden delicious embryos were incubated with (±)-[2-¹⁴C]abscisic acid (ABA) (10^-5 M, 355 kBq mol^-1). After incubations of various durations, the radioactivity was measured in whole embryos, cotyledons, and embryonic axes.With either 48-h or 16-d incubation periods, the uptake of [¹⁴C]ABA depended upon the mode of culture used. The lowest values corresponded to the absorption by the embryonic axis, the highest to the absorption by the distal parts of the two cotyledons. The cotyledons accumulated the main part of the radioactivity under all conditions. Dormant and almost completely after-ripened embryos cultivated for 4 d showed no significant differences in the radioactivity uptake for identical modes of culture. There was a linear relationship between exogenous ABA concentrations (0.5 to 3.10^-5 M) and ABA uptake for embryos cultivated for 4 d with the distal parts of the cotyledons immersed in the medium.Abbreviations ABA abscisic acid. RM, RM⁺, C/2 M, and CM are different modes of embryo cultures: embryonic axis immersed alone (RM), together with the proximal parts of the cotyledons (RM⁺); distal parts of the cotyledons immersed alone (CM); embroyo flat on the medium, the root and the external surface of one cotyledon being in contact with the medium (C/2 M) - PP proximal parts of the cotyledons - DP distal parts of the cotyledons     

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.     

Levels of gibberellin-like substances and their possible transport in developing dwarf and normal cultivars of pea (Pisum sativum L.)

The level of gibberellin-like substances was determined in the cotyledons and axis of developing seedlings of dwarf (Little Marvel) and normal (Tall Telephone) cultivars of pea (Pisum sativum L.). The effect of cotyledon removal with GA₃ application on growth was also examined. Greater levels of gibberellin-like substances were observed in the cotyledons of the normal cultivar than the dwarf. This was particularly evident in the cotyledons during the early stages of seedling growth. Subsequently there was a decline in GA levels in the cotyledons. This was coincidental with a rise in GA content in the axis with markedly greater levels in the normal than the dwarf cultivar. Decotyledonated dwarf and normal plants supplied with GA were much taller than the decotyledonated controls. This observation along with those of the gibberellin levels in the cotyledons and axis, provided circumstantial evidence that there may be translocation of gibberellins from the cotyledons to the axis.     

The Role of ABA in the Control of Apple Seed Dormancy Re-appraised by Combined Gas Chromatography-Mass Spectrometry

Measured by GC—MS²—SIR³, endogenous ABA⁴ in embryonicaxes of seeds of Malus pumila L. cv. Golden Delicious decreased8-fold and cotyledon ABA by only 60%, during 10–50 d ofstratification at 5 ?C, after ABA leaching during an initial24 h soaking. During stratification, the percentage germinationof embryos transferred to 17?C showed a significant linear dependenceon log_e of ABA levels in the axes at transfer. Between 50 and70 d, ABA levels increased markedly in axes and testa both ofstratified seeds and seeds allowed to re-dry at 17 ?C afterinitial soaking. The ability of fully stratified axes with elevatedendogenous ABA to germinate indicated that stratification haddecreased their ABA sensitivity. Changes in cotyledon ABA couldnot account for the promotory effect of cotyledons on germinationduring the first 30 d of stratification. Loss of testa inhibitionof germination during stratification was not linked with changesin testa ABA. Stratification markedly increased the sequestrationin the axes of exogenous ABA supplied via the cotyledons. Changesboth in axis ABA levels and sensitivity were thus correlatedwith dormancy release, but subject to modifying control by thecotyledons and testa not involving ABA. Rehydration of driedseeds affected axis ABA later during storage via mechanismsunconnected with dormancy. Key words: ABA, seed dormancy, stratification     

The importance of root carbohydrate abundance in ammonium uptake

The influence of carbohydrates on ammonium uptake and ammonium transporter (AMT1) expression was investigated in roots of field pea (Pisum arvense) and rutabaga (Brassica napus var. rapifera). Ammonium transport into field pea seedlings diminished markedly following cotyledon removal, which indicated that uptake of ammonium was under control of reserves stored in the cotyledons. Excision of cotyledons decreased also the level of some amino acids, glucose and total reducing sugars in field pea roots. To investigate the importance of the sugar supply for the regulation of ammonium uptake at low external NH ₄ ⁺ level, 1 mM glucose or sucrose was supplied for several hours to the field pea seedlings deprived cotyledons or to intact rutabaga plants. Supply of both sugars resulted in a substantial increase in ammonium uptake by both plant species and enhanced markedly the expression of AMT1 in rutabaga roots. The results indicate that sugars may regulate ammonium transport at the genetic level.     

Microautoradiographic studies of the role of mesophyll and bundle tissues of the Ricinus cotyledon in sucrose uptake

The aim of the study was to show which tissues and cell types of the cotyledon of Ricinus communis L. are responsible for uptake of sucrose by H⁺-sucrose symport. The cotyledons were incubated in labelled sucrose for up to 20 min and then the amount of radioactivity in each cell type of the cotyledon was assessed by microautoradiography. It was found that 50% of the label was present in the spongy mesophyll, and 10–15% was in the bundles, the epidermal layers and the palisade parenchyma. The sieve tubes contained only 2–3% of the label. The addition of sucrose to cotyledons depolarized the membrane of spongy-mesophyll cells by 33 mV. Therefore, it was concluded that the previously found H⁺-sucrose symport is at least partly located at the spongy mesophyll. No precursor-like behaviour of the label in mesophyll or bundle-sheath cells was observed in pulse-chase experiments, which indicates a direct uptake of sucrose by the sieve tube-companion cell complex from the apoplast.This work was funded by Deutsche Forschungsgemeinschaft. The valuable help by Ina Möller, Elke Schmidt, Christian Schobert (all from Bayreuth, FRG), Dr. Dieter Gradmann (Göttingen, FRG), Dr. Jörg Tittor (MPI Biochemie München, FRG), Dr. Pavlovkin (Plant Pathology, Bratislava), Dr. K. Köhler (Botany Department Würzburg, FRG) and the intense discussions with Dr. Enno Brinkmann (Bayreuth) are gratefully acknowledged. The technical assistance by Beatrix Tannhäuser-Hofmann and Hildegard Stork was of great help for this work.     

Is the wild oat embryo monocotylous?

The embryogeny of the wild oat (Avena fatua L.) was studied in detail. The pattern of embryo development was observed to be similar to the other investigated grass taxa, conforming to thePoa variation of the Asterad type. The embryogeny and anatomy of young seedlings showed that the embryo of the wild oat was not monocotylous, but dicotylous. The scutellum of the embryo, as reported for other grasses, was regarded as the first cotyledon, and the first leaf primordium, which developed later into a photosynthesizing leaf and situated opposite the scutellum, was interpreted as the second cotyledon. Observations indicated that the cotyledons of the embryo were placed lateral to the shoot apical meristem, which was terminal in position. The cotyledons were found to be dimorphic in structure and function. The scutellum, a modified cotyledon, functioned as a suctorial organ, transporting nutrients from the endosperm to the embryo axis. The second cotyledon or the first true leaf supplied nutrients directly to the embryo axis through the process of photosynthesis.     

Effect of NaCl and polyethylene glycol on solute content and glycosidase activities during germination of lentil seeds

Abstract Lentil seeds were sown in water and with different concentrations of polyethylene glycol (PEG) 4000 or NaCl. Radicle emergence and growth were delayed by these substances. In cotyledons under stress no variations in solutes occurred, whereas in embryonic axes an accumulation of soluble sugars was observed. The major constituents of the soluble carbohydrates were sucrose, galactose and mannose. Glycosidase activities were not significantly affected in PEG- or NaCl-germinatcd seeds, except axis α-galactosidase, whose activity during axis growth was higher under stress. Water and salt stress did not have a marked effect on carbohydrate metabolism in intact seedlings. The rate of release of ¹⁴C from [6-¹⁴C]-glucose was similar in the stressed seeds and in the control seeds.     

Acyl Lipid Metabolism in Germinating Hazel Seeds (Corylus avellana L.)

A decreasing percentage of radioactivity from [U-¹⁴C]-oleateand [U-¹⁴C]-palmitate was recovered in the lipid fractions ofgerminating hazel cotyledons. The pattern of incorporation ofthe acids into the cotyledon glycerides was consistent withtheir degree of saturation. The relative incorporation intothe cotyledon phospholipids changed during germination. Radioactivityfrom both acids was recovered in increasingly unsaturated fattyacids in the cotyledon hpids. Diversion of both [U-¹⁴C]-fatty acids into acyl lipid synthesisoccurred in the germinating embryonic axes. Both were increasinglyrecovered in mixed glyceride groups which contained diglyceridesand highly unsaturated triglycerides. The two acids gave differentpatterns of incorporation in the axis phospholipids. Desaturationof the acids occurred to a lesser extent than in the germinatingcotyledons.     

Mobilisation of the major stored reserves in the embryo of fenugreek (Trigonella foenum-graecum L., Leguminosae), and correlated enzyme activities

Changes in total nitrogen, soluble amino nitrogen, lipid and phytate contents, and in the activities of proteinase (pH 7.0), isocitrate lyase and phytase were followed in the endosperm, cotyledons, and axis during germination of fenugreek seeds and subsequent growth of the seedlings. The endosperm is comprised largely of cell-wall galactomannans: the majority of the seed total nitrogen, lipid and phytate (5%, 8%, 0.44% of seed dry weight respectively) is localised within the cotyledons as stored reserves. Germination is completed after 10–14 h from the start of imbibition, but the major reserves are not mobilised during the first 24 h. Then the total nitrogen content of the cotyledons starts to decrease and that of the axis increases; there is a concomitant accumulation of soluble amino nitrogen in both cotyledons and axis. An increase in proteinase activity in the cotyledons correlates well with the depletion of total nitrogen therein. Depletion of lipid and phytate reserves in the different seed tissues constitutes a late event, occurring after 50 h from the start of imbibition, and is coincident with the final disintegration of the endosperm tissue. The depletion of phytate and stored lipids is accompanied by an increase in phytase and isocitrate lyase activity. It appears that the products of lipid hydrolysis are converted by gluconeogenesis to serve as the major source of sugars for the growing axis after the endosperm galactomannan has been completely mobilised.     

Degradation of the endosperm cell walls of Lactuca sativa L., cv. grand rapids in relation to the mobilisation of proteins and the production of hydrolytic enzymes in the axis,cotyledons and endosperm

The timing of changes in total nitrogen and soluble amino nitrogen content, and in the activities of proteinase (pH 7.0), isocitrate lyase, catalase, phytase, phosphatase (pH 5.0), -galactosidase and -mannosidase were studied in extracts from the cotyledons, axis and endosperms of germinating and germinated light-promoted lettuce seeds. The largest amount of total nitrogen (2.7% seed dry weight) occurs within the cotyledons, as storage protein. As this decreases the total nitrogen content of the axis increases and the soluble amino nitrogen in the cotyledons and axis increases. Proteinase activity in the cotyledons increases coincidentally with the depletion of total nitrogen therein. Enzymes for phytate mobilisation and for gluconeogenesis of hydrolysed lipids increase in activity in the cotyledons as the appropriate stored reserves decline. Beta-mannosidase, an enzyme involved in the hydrolysis of oligo-mannans released by the action of endo--mannase on mannan reserves in the endosperm, arises within the cotyledons. This indicates that complete hydrolysis of mannans to the monomer does not occur within the endosperm. Mobilisation of all cotyledon reserves occurs after the endosperm has been degraded, providing further evidence that the endosperm is an early source of food reserves for the growing embryo.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid - EDTA ethylenediamine tetraacetic acid disodium salt - TCA trichloroacetic acid Part 2 of a series, of which the first was published in Planta 139, 1–8 (1978)     

Ultrastructural and biochemical investigations of protein mobilization of Mucuna pruriens (L.) DC. cotyledons and embryo axis

The mobilization of storage reserves, with particular emphasis on storage proteins of Mucuna pruriens (L.) DC., cotyledons, and embryo was investigated from the ultrastructural and biochemical points of view. Proteins and starch were the two main storage substances in cotyledons, and proteins and lipids were the main ones in the embryo. Embryo protein bodies were smaller and fewer in number than those of cotyledons. Structural and ultrastructural data determined between 24 and 48 h after imbibition and between 48 and 72 h after imbibition, the end of significant embryo and cotyledon protein mobilization, respectively, indicating more precocious storage protein mobilization in the axis than cotyledons. Moreover, storage protein mobilization in embryo and cotyledons occurred before the end of germination. Water soluble proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, producing 29 bands with molecular weights from 14 to 90 KDa. Embryo extract contained more proteins than cotyledon extract, contained seven characteristic bands, and showed a higher variability of the optical density trend than cotyledon.     

Influence of the Embryonic Axis on Protein Hydrolysis in Cotyledons of Cucurbita maxima

Four-day time course studies of the hydrolysis of cotyledonal storage protein were conducted on intact seeds, seed cotyledons detached from their embryonic axes and on detached cotyledon pairs germinated in the presence of three excised embryonic axes of Cucurbita maxima Duch., cv. Chicago Worted Hubbard. Detached cotyledons germinated alone showed little hydrolysis of the storage protein. However, the amount of protein hydrolysis of the detached cotyledon pairs germinated in the presence of three excised embryonic axes was comparable to the amount hydrolyzed in the cotyledons of intact germinating seeds. Visual growth differences among these treatments were also evident. The size and yellow color intensity of the fourth day treatments were shown to increase in the following order: detached cotyledon pairs alone, intact seedlings, detached cotyledon pairs in the presence of three excised axes. The growth of the hypocotyl and radical was also modified by removal of the cotyledons. These findings suggest that storage protein degradation and cotyledonal growth are controled by the axis. They also indicate that the cotyledons have some influence on the growth of the axes. Time-course studies were made on the hydrolysis of storage protein in the cotyledons of squash and on the distribution of the hydrolytic products during the germination of light- and dark-grown plants. The storage protein was not hydrolyzed during the first 24 hours. It was hydrolyzed at a uniform rate from 1 to 5 days and at a slightly decreased rate from 5 to 7 days. Most of the hydrolytic products were transported to the axial tissue. Proteinase activity in the cotyledons rapidly increased during germination to a maximum level at 2 to 3 days. This was followed by a decline to about the initial value after 7 days.     

Shoot Regeneration from Immature Cotyledons of Cicer Arietinum

Shoot regeneration was achieved from immature cotyledons of five chickpea (Cicer arietinum L.) genotypes: C235, ICC4971, ICC11531, ICC12257 and ICC12873. The cotyledons cultured on Murashige and Skoog (MS) medium supplemented with 3 or 5 mg dm^–3 zeatin with or without 0.04 mg dm^–3 indole acetic acid (IAA) showed formation of cotyledon like structures (CLS) at their proximal ends. Subsequently, shoot regeneration took place in some of the CLS forming explants. CLS were also formed in cotyledons cultured on MS + 0.2 – 1 mg dm^–3 thidiazuron (TDZ); direct shoot regeneration was observed in cotyledons cultured on 1 mg dm^–3 TDZ. The shoot buds elongated on media containing indole butyric acid (IBA), benzylaminopurine (BAP) and gibberellic acid (GA₃). Complete plantlets were obtained by rooting of shoots following pulse treatment with 200 mg dm^–3 IBA for 5 min and culture on growth regulator free half-strength MS medium.     

In vitro plantlet regeneration from cotyledons of the tree-legume Leucaena leucocephala

Two plant regeneration methods applicable to Leucaenaleucocephala were developed. In the first method, involvingorganogenesis via callus formation, cotyledon, hypocotyl and root segments wereinitiated on MS medium containing different concentrations ofN⁶-benzyladenine (BA), 2,4-dichlorophenoxyacetic acid (2,4-D), andnaphthaleneacetic acid (NAA). Both compact (type I) and friable (type II) calliwere obtained from the cotyledon and hypocotyl explants treated with differentconcentrations of the growth regulators. Shoots were generated only from thefriable calli formed from the cotyledon explants. The calli formed from thehypocotyl explants did not generate shoots and the root explants died withoutforming callus. Cotyledon explants from 3–4 day old seedlings showedmaximum callus induction compared to those from older seedlings. In a secondmethod involving direct organogenesis, excised cotyledons were cultured on 1/2MS medium containing 10–35 mg l^–1N⁶-benzyladenine (BA) for 7–14 days. Transfer of thecotyledonsto regeneration medium containing low BA resulted in callus formation andsubsequent shoot regeneration from the base of the excised cotyledon explants,with up to 100% frequency. Regenerated shoots rooted best on a basal mediumcontaining no growth regulators.     

黄皮种子线粒体呼吸速率和活性氧清除酶活性对脱水的响应及其生态学意义

顽拗性种子脱落时具有较高的含水量和代谢活性, 对脱水高度敏感; 但顽拗性种子脱水敏感性的机理至今仍然不清楚。该文以顽拗性黄皮(Clausena lansium)种子为材料, 研究了种子和胚轴对水分丧失的响应, 在脱水过程中胚轴和子叶的呼吸速率, 胚轴和子叶线粒体的细胞色素c氧化酶(CCO)活性、外膜完整性、CCO和交替氧化酶(AOX)途径以及线粒体活性氧清除酶活性的变化。结果表明, 随着水分的丧失, 种子和胚轴的存活率逐渐下降, 种子的脱水敏感性大于胚轴; 胚轴和子叶的呼吸速率以及线粒体外膜的完整性降低。胚轴和子叶线粒体的CCO途径以及胚轴AOX途径的呼吸速率在脱水初期增加, 随着继续脱水下降, 胚轴线粒体AOX途径的呼吸速率则随着脱水显著下降。胚轴线粒体的超氧化物歧化酶(SOD)、抗坏血酸过氧化物酶(APX)和谷胱甘肽还原酶(GR)活性和子叶线粒体的APX活性随着脱水迅速下降; 胚轴线粒体的脱氢抗坏血酸还原酶(DHAR)活性和子叶线粒体的SOD、DHAR和GR活性在脱水初期增加, 然后下降。这些数据表明黄皮种子的脱水敏感性与线粒体的呼吸速率和活性氧清除酶的活性降低密切相关, 也与长期适应热带/亚热带的生境有关。     

Transport and Metabolism of Dihydrozeatin Riboside in Germinating Lupin Seeds

[³H]-dihydrozeatin riboside was applied selectively to the embryonicaxes or to the cotyledons of germinating lupin (Lupinus luteusL. cv. Weiko III) seeds 6 h following the start of imbibition.There was little transport of dihydrozeatin riboside from embryoto cotyledons up to 6 h after the application, but a substantialamount of radioactivity had moved into the cotyledons at theend of the 10 h incubation period. However, there was no detectablemovement of [³H]-dihydrozeatin riboside from the cotyledonsto the embryonic axis. This indicated a highly polarized movementof cytokinins during the early stages of seed germination. Exogenouslyapplied [³H]-dihydrozeatin riboside was found to be very stable,both when applied to the embryonic axes and cotyledons of intactseed, or following excision, and there was little metabolismwith only small amounts of radioactivity found associated withdegradative metabolites. The embryonic axis of this specieshas recently been found to synthesize cytokinins within 12 hfrom the start of imbibition, and the results of this studyindicate that the embryo-derived cytokinin is probably transportedto the cotyledons where it accumulates and subsequently participatesin the control of cotyledon function. Key words: Lupinus luteus, cytokinin transport and metabolism, dihydrozeatin riboside, seed germination     

Isolation and cell regeneration of protoplasts from sugar pine (Pinus lambertiana)

Protoplasts were isolated at high yields from actively growing callus and cell suspensions of cotyledons and needles of mature trees. The best protoplast growth response was obtained from cell suspensions of cotyledon and needle callus. Lower protoplast yields were obtained directly from young needles of flushing buds on explants from mature shoots (30-year-old trees) growing in vitro. In all cases, the first divisions, promoted by dimethyl sulfoxide, were observed in 10–45% of the protoplasts by 7–10 days. After 25–30 days, colonies of 8–10 cells were established. Browning of protoplast-derived cell cultures was observed within 40–45 days (cotyledons) and 20–25 days (mature tree sources).Abbreviations BA N⁶-Benzyladenine - DCR Douglas-fir cotyledon revised medium - 2,4-D 2,4-dichlorophenoxyacetic acid - DMSO dimethyl sulfoxide - FDA Fluorescein diacetate - Mes 2-(N-morpholino) ethanesulfonic acid - NAA -naphthaleneacetic acid