Polypeptide profiles from cotyledons of developing and photoperiodically induced seedlings of the Japanese morning glory (Pharbitis [ipomoea] nil)

An unknown substance(s) produced in the cotyledons of seedlings of the Japanese morning glory (Pharbitis nil) during a defined period of darkness triggers the subsequent initiation of floral buds at apical and axillary meristems. Recent studies have concentrated on characterizing molecular changes as a possible mechanism associated with its synthesis, but these have failed to eliminate interference due to lack of development unity in the sampled population and to consider different kinetic alternatives of those potential changes. The current study demonstrates that numerous age-related changes occur in polypeptides from cotyledons during growth under noninductive conditions, but that these are minimal in older seedlings selected for improved synchrony of the floral response. Polypeptides from older seedlings sampled at various times during and after a dark inductive period were examined by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). No differences associated with floral induction could be detected. The results indicate that changes in gene expression correlated with floral-induction do not occur in the abundant polypeptide fraction within the limitations of the techniques employed.     

Enzymatic changes in gourd and bean cotyledons during ageing and the effect of detopping

Cotyledons of gourd (Cucurbita maxima Duchesne) and bean (Phaseolus vulgaris L.) were used to study the changes in the activities of catalase, peroxidase, acid inorganic pyrophosphatase and alkaline inorganic pyrophosphatase during ageing and the diversion in such changes that occur when cotyledon senescence was retarded by detopping the seedlings above the cotyledons. Catalase, acid inorganic pyrophosphatase and alkaline inorganic pyrophosphatase activities declined during the senescence of the cotyledons. When cotyledon senescence was retarded by detopping as marked by the increase in the levels of chlorophyll and protein, there was also an increase in the activities of these enzymes. Peroxidase activity, on the other hand, increased during the senescence of the cotyledons and detopping the seedlings resulted in a further increase in the peroxidase activity. It can be suggested that some root factor(s) probably cytokinin(s) is (are) mobilised into the cotyledons of the detopped seedlings which otherwise would have been mobilised into the shoot apices, and help retard or even reverse the senescence of the cotyledons.     

Role of ethylene in chlorophyll degradation in radish cotyledons

The effect of age of radish seedlings on changes in chlorophyll concentration caused by ethylene was examined. Ethylene was produced at 2–4 nl g^?1 h^?1 following excision of cotyledons from 5-to 20-day-old seedlings. The youngest cotyledons maintained this rate, whereas ethylene synthesis declined by as much as 80% during a 24-h period in older cotyledons. The youngest cotyledons continued to accumulate chlorophyll in the dark, but after 7 days cotyledons lost chlorophyll and the proportion of chlorophyll lost increased with age. Ethylene promoted, and norbornadiene inhibited, this loss of chlorophyll; in combined treatments the effects of ethylene and norbornadiene were competitive. The maximal rate of chlorophyll loss occurred in 1μl L^?1 ethylene; extrapolation of the response to concentration indicated that half-maximum loss would occur at 0.005–0.01 μl L^?1 ethylene. In cotyledons from 20-day-old seedlings, chlorophyll degradation occurred mainly after 24 h from excision and transfer to the dark. Chlorophyll degradation during 48 h in the dark was affected by norbornadiene or ethylene applied from 0–24 h or from 24–48 h.     

Stimulation of the Flowering of Pharbitis nil Chois. by Gibberellin A3 : Time Dependent Action at the Apex

Gibberellin A₃ (GA₃) stimulated flowering when it was appliedto the shoot apex of seedlings of Pharbitis nil, dwarf strainKidachi; but, not when it was applied to the cotyledons. GA₃applied to the plumule before or shortly after the start ofan inductive dark period promoted both flowering and shoot elongation;but, the later the time of application during the dark periodless the promotion of flowering, although marked promotion ofshoot elongation always took place. The variation with time in the response of flowering to GA₃indicates that early floral processes at the apex are stimulatedby GA₃, but that subsequent processes are insensitive to it.The early processes of floral stimulus produced by a 16 hr inductivedark period probably are completed within 20 hr at 28°Cafter the end of the dark period. At low temperatures, suchas 15 and 20°C, early floral processes continued for morethan 40 hr. When cotyledons were removed at various times, the export ofthe floral stimulus to the shoot apex was apparent within hoursof the generation of the floral stimulus in the cotyledons,which started with the passage of the critical 9-hr dark period. (Received February 18, 1981; Accepted March 24, 1981)     

The Role of Cotyledons in Flower Initiation of Pharbitis nil at Low Temperatures

Seedlings of Pharbitis nil, Strains Violet, Tendan and Kidachi,initiated floral buds under Continuous light when exposed totemperatures lower than 15, 15 and 21?C, respectively, throughoutthe experimental period, or to 13–14?C for a minimum durationof 10, 8 and 4 days, respectively. Cotyledons were necessaryfor floral initiation when the seedlings at the start of coldtreatment were 8 days old (10 days old for Kidachi) or younger,although neither cotyledons nor foliage leaves were necessarywhen the plants were older. When the cotyledons in young seedlingswere removed immediately after exposure to cold temperature(13–14?C) for 14 (Violet), 12 (Tendan) or 8 (Kidachi)days (cold treatment begun when the cotyledons had just unfolded),only a few plants initiated floral buds under continuous light.However, when the cotyledons were left attached for 2 more daysat 23?C, the plants produced as many flower buds as those withintact cotyledons, suggesting that cotyledons exposed to coldtemperature produce a floral stimulus which can be translocatedto buds even after the end of the cold treatment. (Received October 14, 1981; Accepted January 20, 1982)     

Identification of nucleases related to nutrient mobilization in senescing cotyledons from French bean

The knowledge about the physiological function of plant nucleases is scarce besides that they have been involved in nucleic acid degradation related with programmed cell death processes. Cotyledons provide a suitable system to investigate this process and the changes associated to nutrient mobilization. Nuclease activities have been determined in French bean seedlings. The total nuclease activity in French bean cotyledons is lower than in embryonic axes; however, several nucleases were detected by in-gel nuclease activity assays with extracts from cotyledons of French bean and ssDNA as substrate. The nuclease activities induced during cotyledon senescence showed higher activity at neutral than at acidic pH. Five different nuclease genes belonging to S1/P1 family have been identified in French bean genome database named PVN1 to PVN5. Their relative expression in cotyledons has been determined from the start of imbibition to senescence, and three genes from this family showed expression in cotyledons. PVN1 was expressed during early stages of seedlings development, whereas PVN4 and PVN5 were expressed during cotyledons senescence. The removal of epicotyl in French bean seedlings resulted in a decrease in the activity and in the expression of the genes associated with the cotyledons senescence process, i.e. PVN4 and PVN5. At the same time, the mobilization of reserves in those cotyledons was slowed down. In the same way, the deficit in phosphate and nitrate during seedlings development led to an acceleration of induction of these genes at the same time that reserves were utilized early on the time. Therefore, the induction of PVN4 and PVN5, the two S1 nuclease genes involved in the process of cotyledon senescence, is related to nutrient mobilization, supporting a possible role for nucleic acids in nutrient recycling during cotyledon senescence.     

Role of ethylene in chlorophyll degradation in radish cotyledons

The effect of age of radish seedlings on changes in chlorophyll concentration caused by ethylene was examined. Ethylene was produced at 2–4 nl g^–1 h^–1 following excision of cotyledons from 5-to 20-day-old seedlings. The youngest cotyledons maintained this rate, whereas ethylene synthesis declined by as much as 80% during a 24-h period in older cotyledons. The youngest cotyledons continued to accumulate chlorophyll in the dark, but after 7 days cotyledons lost chlorophyll and the proportion of chlorophyll lost increased with age. Ethylene promoted, and norbornadiene inhibited, this loss of chlorophyll; in combined treatments the effects of ethylene and norbornadiene were competitive. The maximal rate of chlorophyll loss occurred in 1l L^–1 ethylene; extrapolation of the response to concentration indicated that half-maximum loss would occur at 0.005–0.01 l L^–1 ethylene. In cotyledons from 20-day-old seedlings, chlorophyll degradation occurred mainly after 24 h from excision and transfer to the dark. Chlorophyll degradation during 48 h in the dark was affected by norbornadiene or ethylene applied from 0–24 h or from 24–48 h.     

In situ fossil seedlings of a Metasequoia-like taxodiaceous conifer from Paleocene river floodplain deposits of central Alberta,Canada

Fossil seeds and seedlings of a Metasequoia-like taxodiaceous conifer occur in Paleocene deposits at the Munce's Hill and Gao Mine localities of central Alberta, Canada. Compression/impression specimens are preserved in upright growth positions among seedlings of the cercidiphyllaceous dicot Joffrea speirsii Crane & Stockey. There are a large number of seeds, a few of which were buried while germinating and show a radicle or short primary root. More than 500 Metasequoia-like seedlings have been identified that have two linear cotyledons with parallel margins and rounded tips. Three specimens have been found that display three cotyledons. Slightly older seedlings show decussate pairs of leaves attached to the stem distal to the cotyledons. Still older seedlings have axillary branches that show varying sizes and numbers of opposite leaves arranged in a single plane distal to the opposite pairs. These specimens reveal that both Joffrea and this extinct taxodiaceous conifer were early colonizers of North American floodplain communities at the beginning of the Tertiary.     

Changes in Cotyledon mRNA during Ethylene Inhibition of Floral Induction in Pharbitis nil Strain Violet

Floral induction in seedlings of Pharbitis nil strain Violet, with one cotyledon removed, was manipulated by applying various ethylene treatments to the remaining cotyledon during a 16 hour inductive dark period. Exposure of cotyledons to ethylene (100 microliters per liter) for 4 hours at different times during the dark period inhibited flowering to some extent, with inhibition being greater towards the end of the dark period. RNA from cotyledons given a 16 hour dark period (induced) or exposed to 100 microliters per liter ethylene throughout the dark period, which completely inhibited flowering, was examined. The poly(A)⁺RNA was translated in vitro using a wheat germ system, and the resulting translation products were analyzed by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. There were substantial qualitative and quantitative differences between the poly(A)⁺RNA extracted from induced cotyledons and that from those exposed to ethylene throughout the dark period. Some of these changes are similar to those observed when flowering was inhibited by photoperiodic treatments (M Lay-Yee, RM Sachs, MS Reid 1987 Planta. In press). The significance of these findings to our understanding of the molecular control of flower induction is discussed.     

Control of phenylalanine ammonia-lyase and cinnamic acid 4-hydroxylase in gherkin tissues

Blue light mediates a transient increase in the extractable activity of phenylalanine ammonia-lyase from both cotyledons and hypocotyls of etiolated gherkin seedlings, but concurrent changes in extractable cinnamic acid 4-hydroxylase activity only occur in cotyledons. Excision, followed by incubation in the dark, also results in stimulation of the lyase activity in both tissues, but the hydroxylase activity is only stimulated in cotyledons, again concurrently with the lyase. Stimulated levels of hydroxycinnamic acid esters are, however, only formed following light treatment, indicating the presence of another light-sensitive step in their biosynthesis. Treatment of gherkin tissues with 2-aminooxyacetic acid or α-aminooxy-β-phenylpropionic acid inhibits phenylalanine ammonia-lyase activity in situ, reduces the accumulation of hydroxycinnamic acid esters and presumably reduces the endogenous cinnamic acid pool. This treatment increases extractable lyase activity and delays its peak in activity. In cotyledons, these changes in the lyase are associated with concurrent and similar changes in extractable hydroxylase activity, whilst in hypocotyls the hydroxylase is relatively unaffacted. The increase in phenylalanine ammonia-lyase activity following excision of cotyledons and hypocotyls is prevented by cinnamic acid; in cotyledons the hydroxylase is similarly affected, but after a longer lag. Thus whilst cinnamic acid can modify the extractable activity of the lyase, it cannot itself mediate changes in the extractable activity of the hydroxylase.     

Increased Na+ and Cl− accumulation induced by NaCl salinity inhibits cotyledonary reserve mobilization and alters the source-sink relationship in establishing dwarf cashew seedlings

We studied the NaCl-induced changes in cotyledons and the embryonic axis of establishing dwarf cashew (Anacardium occidentale) seedlings. The salt stress reduced the growth of dwarf cashew seedlings, and this response was related to the inhibition of cotyledonary reserve depletion. Lipid mobilization was inhibited by NaCl due to reduced lipase activity in the emerging and establishing seedlings. Additionally, there was reduced transient starch accumulation in the cotyledons of the salt-stressed seedlings that was associated with lower starch synthase activity at the early developmental stages and inhibited amylolytic and starch phosphorylase activities at the established seedling stage. The NaCl-induced changes in lipid and starch metabolism influenced the soluble sugar content in the cotyledons. Protein mobilization was inhibited by NaCl, and we observed the accumulation of amino acids and the inhibition of proteolytic activity in the cotyledons of the salt-stressed established seedlings. Salinity significantly reduced the free amino acid and reducing sugar contents in the embryonic axes of both emerged and established seedlings, whereas the non-reducing sugar content was affected by this stress only in the established seedlings. The Na⁺ and Cl^? contents progressively increased in the cotyledons and embryonic axis of the seedlings as the salinity increased. We conclude that salt stress inhibits dwarf cashew seedling establishment by inhibiting the mobilization of reserves, an inhibition that was related to increased Na⁺ and Cl^? accumulation in the cotyledons. Additionally, these toxic ions reduced the sink strength of the embryonic axis with regard to the products of cotyledonary reserve mobilization.     

Changes in Glycolipid and Phospholipid Compositions in Cotyledons of Germinating Soybeans

This investigation was conducted to observe changes in the compositions of fatty acids, glycolipids (GL) and phospholipids (PL) in cotyledons of soybean seeds which were germinated either in the dark or the light at 28°C for 8 days. The patterns of changes in lipid composition depended on the germinating conditions tested. In general, non-polar lipids were metabolized at a faster rate than polar lipids. Changes in lipid contents in cotyledons were also observed more clearly with the polar lipids than with the non-polar ones, especially in the light-grown seedlings. The major component of lipid, GL in chloroplasts, appeared rapidly at an earlier stage in the cotyledons of light-grown seedlings. During germination of soybean seeds, acyl sterylglucoside in cotyledons decreased rapidly, but monogalactosyl diglyceride and digalactosyl diglyceride (DGD) increased in the light-grown seedlings, whereas sterylglucoside and DGD increased in the dark-grown seedlings.

The major PL present immediately after immersion were phosphatidyl ethanolamine (PE), phosphatidyl choline (PC) and phosphatidyl inositol (PI). During germination under both conditions, light and dark, PE in cotyledons decreased with PC or PI, while phosphatidic acid increased rapidly, and phosphatidyl glycerol and diphosphatidyl glycerol also increased slightly. These changes in glycolipid and phospholipid compositions during germination seem to occur from the formation of photosynthetic tissues and the metabolic interconversion of phospholipids.     

Purine synthesis and catabolism in soybean seedlings : the biogenesis of ureides

The ureides, allantoin and allantoic acid, are the major nitrogenous substances transported within the xylem of N₂-fixing soybeans (Glycine max L. Merr. cv Amsoy 71). The ureides accumulated in the cotyledons, roots and shoots of soybean seedlings inoculated with Rhizobium or grown in the presence of 10 millimolar nitrate. The patterns of activity for uricase and allantoinase, enzymes involved in ureide synthesis, were positively correlated with the accumulation of ureides in the roots and cotyledons. Allopurinol and azaserine inhibited ureide production in 3-day-old cotyledons while no inhibition was observed in the roots. Incubation of 4-day-old seedlings with [¹⁴C]serine indicated that in the cotyledons ureides arose via de novo synthesis of purines. The source of ureides in both 3- and 4-day-old roots was probably the cotyledons. The inhibition of ureide accumulation by allopurinol but not azaserine in 8-day-old cotyledons suggested that ureides in these older cotyledons arose via nucleotide breakdown. Incubation of 8-day-old plants with [¹⁴C]serine suggested that the roots had acquired the capability to synthesize ureides via de novo synthesis of purines. These data indicate that both de novo purine synthesis and nucleotide breakdown are involved in the production of ureides in young soybean seedlings.     

Indirect Action of Benzyladenine and Other Chemicals on Flowering of Pharbitis nil Chois: Action by Interference with Assimilate Translocation from Induced Cotyledons

Benzyladenine (BA) brushed on the cotyledons of 4-day-old seedlings of Pharbitis nil Chois. markedly stimulates flowering. Greates response is obtained for concentrations between 44 and 440 micromolar. The action of BA is on processes in the cotyledon as shown by the response to its site of application, to the dosage applied and to the requirement for its application prior to the dark period. There was little or no effect of BA treatment on either the time measurement processes of photoperiodic induction or on the generation of floral stimulus. Transport of photosynthetic assimilate from the cotyledons to the shoot apex was altered.     

Seed and soil: sources of inoculum for the colonisation of the foliage of solanaceous hosts by Pseudomonas solanacearum

Pseudomonas solanacearum was transmitted from contaminated seed to the cotyledons of capsicum (Capsicum frutescens) at 92, 73 and 60% r. h. and to the cotyledons of tomato (Lycopersicon esculentum) seedlings at 92% r. h. Subsequent epiphytic colonisation of the true leaves of capsicum occurred at 92 and 73% r. h. An increase in the population on capsicum cotyledons was detected at 92 and 73% r. h. but only at the higher r. h. was an increased population of P. solanacearum associated with the colonisation of the true leaves. Lesions developed on the true leaves of capsicum at 92% r. h. Transmission of P. solanacearum from capsicum seeds was affected by inoculum concentration, occurring at an infestation level of ∽ 10³ propagules seed^-1 but not at 50 propagules seed^-1. Pseudomonas solanacearum was detected on the cotyledons of capsicum held at 98% r. h. after germination of seed in soil infested with 2 × 10⁸ propagules g^-1 soil; lesions were detected on cotyledons 11 days after planting and invasion occurred in 10 stems and one root of the 20 seedlings sampled. The movement of the pathogen from the soil to the seedlings was affected by the level of soil infestation. Pseudomonas solanacearum was detected in four pairs of cotyledons, two stems and one root of the 36 seedlings sampled from soil infested with 10⁷ propagules g^-1 soil but it was not isolated from seedlings sampled from soil infested with 10⁵ or 4 × 10³ propagules g^-1 soil. Leaf and stem prints demonstrated the epiphytic nature of this organism on the cotyledons and stems of seedlings.     

The effect of polyploidy and hybridization on the evolution of floral colour in Nicotiana (Solanaceae)

Background and Aims Speciation in angiosperms can be accompanied by changes in floral colour that may influence pollinator preference and reproductive isolation. This study investigates whether changes in floral colour can accompany polyploid and homoploid hybridization, important processes in angiosperm evolution.Methods Spectral reflectance of corolla tissue was examined for 60 Nicotiana (Solanaceae) accessions (41 taxa) based on spectral shape (corresponding to pigmentation) as well as bee and hummingbird colour perception in order to assess patterns of floral colour evolution. Polyploid and homoploid hybrid spectra were compared with those of their progenitors to evaluate whether hybridization has resulted in floral colour shifts.Key Results Floral colour categories in Nicotiana seem to have arisen multiple times independently during the evolution of the genus. Most younger polyploids displayed an unexpected floral colour, considering those of their progenitors, in the colour perception of at least one pollinator type, whereas older polyploids tended to resemble one or both of their progenitors.Conclusions Floral colour evolution in Nicotiana is weakly constrained by phylogeny, and colour shifts do occur in association with both polyploid and homoploid hybrid divergence. Transgressive floral colour in N. tabacum has arisen by inheritance of anthocyanin pigmentation from its paternal progenitor while having a plastid phenotype like its maternal progenitor. Potentially, floral colour evolution has been driven by, or resulted in, pollinator shifts. However, those polyploids that are not sympatric (on a regional scale) with their progenitor lineages are typically not divergent in floral colour from them, perhaps because of a lack of competition for pollinators.     

Development of the shoot apex ofChenopodium rubrum L. after photoperiodic induction in the cotyledon stage

Development of the shoot apex up to floral differentiation was investigated in the short-day plantChenopodium rubrum. The changes occurring in the apex from energence until full opening of the cotyledons (Figs 1–4), development during photoperiodic induction (Figs. 5–8), as well as the resulting floral differentiation (Figs. 9–10) are described. It was aimed at excluding the influence of plastochron changes on the interpretation of ontogeny of the shoot apex. For that reason two planes of longitudinal sections and two plastochron stages were compared. In young plants zonation does not become fully evident prior to floral differentiation. The anatomical structure of the shoot apex does not change substantially during the first two inductive cycles which proved to be obligatory under the given experimental conditions. The changes occurring during two further inductive cycles correspond to the total activation of the meristems as manifested by the growth and branching of the apex preceeding floral differentiation proper.     

Synthesis of Chloroplast Proteins during Germination and Early Development of Cucumber

Cell-free protein synthesizing systems have been used to study the developmental changes in the synthesis of chloroplast proteins in the cotyledons of cucumber seedlings grown in the light or in the dark. Escherichia coli and wheat germ in vitro protein synthesizing systems have been used to assay the changes in the levels of the mRNA's coding for ribulose 1,5-bisphosphate carboxylase (RuBPCase). The large subunit of cucumber RuBPCase has been identified among the translation products of the E. coli system. The wheat germ system translates the cucumber mRNA coding for the small subunit of RuBPCase to produce a 25,000 molecular weight precursor polypeptide. Plastids isolated from light-grown cotyledons were used to study developmental changes in their capacity to synthesize protein. The data obtained indicate that in the light there is an initial 48-hour period of accumulation of the mRNA's coding for the large and small subunits of RuBPCase, coupled with an increase in the capacity of the isolated plastids to synthesize protein. This is followed by a decline. This decline is not reflected in the accumulation of RuBPCase in the cotyledons which remains constant over the period of study.     

Mitochondrial folates and methionyl-tRNA transformylase activity during germination and early growth of seeds

Mitochondria were isolated from the cotyledons of pea (Pisum sativum cv Homesteader) and peanut (Arachis hypogaea cv Early Spanish) seeds over a 7-day growth period. The rate of mitochondrial oxygen uptake increased 3-4-fold during the first 4 days of growth and parallel changes were observed in the respiratory control and ADP/O ratios. In both species, the total cotyledonary pool of folate derivatives increased 3-4-fold during this period of germination whereas that associated with isolated mitochondria increased 5-10-fold. Until day 3 of growth, the mitochondrial folates were principally polyglutamates of 10-formyltetrahydrofolate but between day 4 and day 7 increasing levels of 5-methyltetrahydrofolate polyglutamates were detected. Pea and peanut mitochondria contained methionyl-tRNA transformylase (EC 2.1.2.9) activity that displayed an absolute requirement for 10-formyl-tetrahydrofolate. The specific activity of this enzyme rose during germination, reaching maximal levels between days 3 and 4. Isolated pea mitochondria had the ability to incorporate [³H]leucine and [³⁵S]methionine into protein in a reaction that required ADP and malate but was strongly inhibited by chloramphenicol. Organelles isolated after 4 days of germination incorporated leucine at rates ca 5-fold greater than shown by mitochondria of 16-hour-old seedlings. The inter-relationships between respiratory activity, mitochondrial formyltetrahydrofolates and methionyl-tRNA transformylase activity suggest a role for organelle protein synthesis during germination of these legume species.