Effects of Sugars on Ethylene Production by Excised Sinapis Cotyledons

Ethylene production by excised Sinapis cotyledons in the lightwas increased 2–3 fold by exogenous 0–06 M sucroseafter a 6 h lag period and by glucose after a lag of 24–48h, whereas the non-metabolized glucose analogue 3-O-methyl -D-glucopyranosehad no effect on ethylene production. Sucrose enhancement of ethylene production occurred in boththe petiole and lamina of the cotyledon and was greater at highlight intensities than at low intensities, or in the dark. Enhanced ethylene levels in the petiole may be responsible forthe decline in petiolar protein, RNA and chlorophyll, and theregression of root primordia in cotyledons incubated in sucrose.     

Light Effects on the Nucleic Acids of Excised Cotton Cotyledons

The effects of light and glucose in the nutrient medium on the nucleic acid metabolism of excised 8-day cotton (Gossypium hirsutum var. Acala 44) cotyledons were determined. The rates of synthesis as affected by light and glucose were determined by brief exposures to C(14)-labeled orotic acid. The nucleic acids were fractionated by homogenizing in Tris-HCl buffer and centrifuging to obtain soluble and microsomal RNA (20,000 x g supernatant) and a particulate nucleic acid fraction (20,000 x g precipitate) or by extracting in phenol followed by 10% NaCl extraction at 100 degrees . The phenol extract was analyzed by density gradient centrifugation.Light and glucose caused parallel changes in nucleic acid levels of the various fractions, in orotic acid-6-C(14) absorption and in rates of synthesis of nucleic acids. Light and glucose appear to enhance binding of the ribosome nucleic acid so that it becomes less extractable in Tris-HCl buffer or phenol. The bound nucleic acids were labeled at a slightly higher rate than the total nucleic acids extracted by Tris-HCl or phenol. However, light treatment for 48 hours promoted a very high labeling rate in the soluble, low molecular weight nucleic acid as shown by density gradient centrifugation of the phenol extractable fraction.It was concluded that a part of the nucleic acid changes were brought about by light acting through the photosynthetic production of carbohydrate. This conclusion was strengthened by the observation that herbicide inhibitors of photosynthesis and limited atmospheric CO(2) concentrations partially inhibited the nucleic acid changes. However, glucose did not cause changes in nucleic acid levels as large as those caused by light and changes were observed to occur in light even though the endogenous sugar levels were maintained at a low level by the inhibition of photosynthesis with herbicides. The data indicated that light may produce changes in nucleic acid levels by other mechanisms additional to those regulating the sugar level in the tissue.     

Effect of the Hypocotyl Hook on Chlorophyll Accumulation in Excised Cotyledons of Cucumis sativus L

Hypocotyl hooks have been shown to influence greening in excised cucumber (Cucumis sativus) cotyledons. The properties of the lag phase are greatly affected by the presence or absence of the hook tissue. A 45-second light pretreatment followed by 4 hours of darkness is sufficient to remove the lag phase from cotyledons with hooks, while hookless cotyledons require 2 hours of continuous illumination followed by 1 hour of dark incubation to break the lag phase. The effect of hooks on cotyledon greening is enhanced if the hooks are shielded from light. Cutting off the hooks after lag phase removal caused a marked decrease in chlorophyll accumulation in the cotyledons. These observations may indicate that the hypocotyl hooks produce a substance or substances needed in the greening process, which are translocated to the cotyledons. Indoleacetic acid, abscisic acid, gibberellin A₃, 6-benzylamino purine and δ-aminolevulinic acid do not show any activity; on the other hand, ethylene appears to replace partially the hypocotyl hooks.     

The Effect of Sucrose on Primordium Development and on Protein and RNA Levels in Detached Cotyledons of Sinapis alba L.

Cotyledons of Sinapis alba cultured in the light on filter-papermoistened with water initiated root primordia within 2 daysafter detachment. These increased in size and penetrated thebase of the petiole after a further 3 days. Culture in sucrosesolution in the light inhibited root formation mainly by affectingthe extension phase when many primordia regressed resultingin a decline in the numbers of primordia per petiole. When cotyledonswere cultured in the dark no primordia were initiated in water,but sucrose promoted root formation. Levels of RNA rose to a peak value in the terminal petiole segmentat a time when primordia were being initiated in this region,protein reached a peak 2 days later. This pattern was much lessevident in the subterminal segments. It did not occur at allin cotyledons cultured in sucrose in the light where a smallinitial increase in RNA and protein was followed by a rapiddecline. The level of RNA and protein in the terminal segmentin light-cultured cotyledons in water was related to the numberof light periods given following detachment as was the percentageof cotyledons which formed roots. The effect of sucrose on RNAand protein content was less marked when cotyledons were transferredfrom sucrose to water instead of being maintained in sucrosethroughout. Sucrose clearly modifies the biochemical and structural changestaking place at the petiole base having particularly inhibitoryeffects on the extension phase of the primordia. The evidenceindicates that sucrose could be causing premature ageing ofcells forming the primordia.     

Biochemical Mechanism for Regulation of Sucrose Accumulation in Leaves during Photosynthesis

It is not known why some species accumulate high concentrations of sucrose in leaves during photosynthesis while others do not. To determine the possible basis, we have studied 10 species, known to differ in the accumulation of sucrose, in terms of activities of sucrose hydrolyzing enzymes. In general, acid invertase activity decreased as leaves expanded; however, activities remaining in mature, fully expanded leaves ranged from low (<10 micromoles per gram fresh weight per hour) to very high (>100 micromoles per gram fresh weight per hour). In contrast, sucrose synthase activities were low and relatively similar among the species (4-10 micromoles per gram fresh weight per hour). Importantly, leaf sucrose concentration, measured at midafternoon, was negatively correlated with acid invertase activity. We propose that sucrose accumulation in vacuoles of species such as soybean and tobacco is prevented by acid invertase-mediated hydrolysis. Initial attempts were made to characterize the relatively high activity of acid invertase from mature soybean leaves. Two apparent forms of the enzyme were resolved by Mono Q chromatography. The two forms had similar affinity for substrate (apparent K_m [sucrose] = 3 millimolar) and did not interconvert upon rechromatography. It appeared that the loss of whole leaf invertase activity during expansion was largely the result of changes in one of the enzyme forms. Overall, the results provide a mechanism to explain why some species do not accumulate sucrose in their leaves. Some futile cycling between sucrose and hexose sugars is postulated to occur in these species, and thus, the energy cost of sucrose production may be higher than is generally thought.     

Effects of Ear Removal on Photosynthesis, Carbohydrate Accumulation and on the Distribution of Assimilated 14C in Wheat

Removal of an ear from a tiller of a wheat plant growing inthe field did not result in any marked change in the net photosyntheticrate of the subtending flag leaf, even during the period whenthe ear would normally have received large amounts of assimilatefrom the flag leaf. Following ear removal, there was an increasein the amount of ethanol-soluble and ethanol-insoluble carbohydratesin the remaining organs of the tiller. ¹⁴C labelling studiesshowed that a new pattern of translocation was established within2–3 days of ear removal, and the tiller exported assimilateto other tillers on the plant, and possibly to the roots.     

Some Effects of Sucrose Concentration on Primordium Development in Excised Primary Roots

The effects of varying the amount of sucrose used to supplementthe culture medium maintaining the growth of excised roots ofPisum sativum L., Vicia faba L., Zea mays L. and Phaseolus vulgarisL., on the rates of primordium initiation and subsequent emergenceas lateral roots and on the duration of the interval betweenprimordium inception and emergence as a secondary root throughthe tissues of the primary have been investigated. Variation in the exogenous concentration of sucrose from 0.5to 8 per cent had little effect on the rate of primordium inceptionin Pisum and Vicia and the rates never reached the values obtainedfor the roots of the corresponding intact plants. Moreover,over the 6 day culture period lateral root emergence did notoccur in any of the excised roots of these two species. In contrast,each of the aspects of primordium development examined in theexcised roots of Zea and Phaseolus was markedly affected bythe amount of sucrose used to supplement the culture medium.In addition, in the presence of about 6 per cent sucrose, primordiumdevelopment in these cultured roots was very similar to thatin roots of the corresponding intact plants. The results indicate either that some factor necessary for primordiumdevelopment is present in adequate amounts in excised rootsof Zea and Phaseolus, but not in those of Pisum and Vicia, orthat the factors controlling such development are differentin the former and latter two species. Vicia faba L., Pisum sativum L., Zea mays L., Phaseolus vulgaris L., broad bean, garden pea, maize, dwarf bean, primordium development, sucrose concentration, cultured roots     

Effects of Sucrose on Rooting and Senescence in Detached Raphanus sativus L. Cotyledons

Application of 0.5–4.0 per cent sucrose to excised radishcotyledons incubated in the light suppressed the formation ofroots, enhanced the rate of yellowing, and altered the locationof chlorophyll loss from the margin of the lamina to the petiolarregion in most of the cotyledons. These effects were not producedwhen osmotica such as mannitol or polyethylene glycol were used. A few cotyledons were able to root in the presence of sucroseand these showed enhanced yellowing at the margin. The numberof such cotyledons increased when they were incubated in waterfor the period during which primordium initiation begins, beforetransfer to sucrose. However, the pattern of yellowing in thepresence of sucrose was not affected by the repeated removalof the region of primordium development, suggesting that thepattern of senescence is not controlled by the formation ofroot primordia. The results are discussed in relation to the effects of lightand possible accumulation of carbohydrate on senescence androot formation in excised tissues.     

The Effects of Temperature on Root Initiation in Detached Cotyledons of Sinapis alba L.

A thermoplate system is described which enables uniformly illuminatedpetri dish cultures to be maintained under a range of temperatureregimes. The system is evaluated and used to investigate theeffects of incubation temperatures between 10 and 35 ?C on rootinitiation. Sinapis cotyledons root most rapidly and in greatest numbersat 25 and 30 ?C. Below 25 ?C, rooting is reduced and delayed,and above 30 ?C rooting is prevented. The optimum temperaturefor initiation of primordia is close to 25 ?C, where, after4 days over 80 per cent of petioles have at least one primordiumand over half of these are already elongating. Initiation isconsiderably reduced at temperatures above 30 ?C and below 25?C and primordium elongation either fails to take place (35?C) or is delayed (20, 13, 10 ?C). At 25 ?C, 90 per cent of the primordia are initiated in theterminal 1.5 mm of the petriole. A similar pattern occurs atother temperatures, but the relative numbers of primordia initiatedin the upper petiole are increased when cotyledons are incubatedat temperatures below 25 ?C. At 25 ?C a higher proportion ofprimordia are initiated in the upper petiole in cotyledons withmany primordia than in cotyledons with few, but the developmentof primordia to the elongation stage is not affected by theirposition in the petiole nor by the numbers of primordia present.     

The Carbohydrate Nutrition of Tomato Roots: III. The Effects of. External Sucrose Concentration on the Growth and Anatomy of Excised Roots

Variation of the concentration of sucrose in White‘s mediummarkedly affects the increase in length of the main axis, andthe number and total length of the laterals developed by excisedtomato roots. Highest values for these three features are obtainedwith medium containing 1.5 per cent, sucrose. Root materialhas been analysed anatomically. Root diameter, cortical thickness,number of xylem vessels, and development of the cortical air-spacesystem increase with increase in sucrose concentration. Thedegree of vacuolation of extra-stelar meristematic cells increases,and protophloem differentiates further from the promeristemwith rising sucrose concentrations. The interpretation of theseresults is discussed.     

Effects of Ammonium and Potassium Ions on Some Physiological and Biochemical Processes of Excised Cucumber Cotyledons

Incubation of excised cucumber cotyledons (Cucumis sativus L.) with NH₄Cl solutions exceeding 0.001 M inhibited their greening, fresh weight increases, and incorporation of ¹⁴C-leucine into insoluble N compounds. The absorption of ¹⁴C-leucine during incubation and retention of moisture by the excised cotyledons after incubation were greatly diminished by the NH₄Cl treatments. Treatment with KCl solutions of the same concentrations as the NH₄Cl stimulated the greening, fresh weight increases, and the absorption and incorporation of ¹⁴C-leucine. Desiccation of cotyledons stored at 5°C for 10 days was inhibited by dilute KCl solutions. The toxicity of NH₄Cl was alleviated by KCl solutions at low concentrations.     

The Effects of Light Intensity and Sucrose on Root Formation, Photosynthetic Ability, and Senescence in Detached Cotyledons of Sinapis alba L. and Raphanus sativus L.

The ability of detached cotyledons cultured in the light toassimilate ¹⁴CO₂, was reduced by the presence of sucrose inthe culture medium. This was due, at least in part, to an increasedrate of chlorophyll loss and yellowing of the blade. When cotyledondiscs were used, the inhibition of ¹⁴Carbon fixation by sucrosewas even more marked than in entire cotyledons. This could bedue to a higher level of penetration of the sucrose into discsor to the absence of the petiole which normally accumulatesphotosynthetic products. Sucrose culture also inhibited root production in cotyledonscultured in the light but promoted root formation in dark-grownor DCMU-treated cotyledons. The DCMU-inhibition of ¹⁴Carbonfixation by the blades was alleviated to some extent by sucroseculture. The sucrose effect on rooting was not permanent inthat transfer into water from sucrose led to root formationalthough this was delayed and present in a lower proportionof cotyledons than the controls. Thus, although a carbohydrate source either from photosynthesisor as applied sucrose, is essential for root production to takeplace, the combination of culture in the light with the presenceof sucrose in the medium may lead to an accumulation of carbohydrateto a level which directly or indirectly increases blade yellowingand inhibits root production.     

The Effects of Age and Root Formation on the Life-Span of Excised Cotyledons of Cucurbita pepo

Root initiation in Cucurbita cotyledons excised at various ages was studied in nutrient solution and water. The time required for root initiation depends on the age of the cotyledon and on the rooting medium. Nutrient solution favoured root formation. Maximum number of roots, maximal total root length, and maximum life-span were found with cotyledons excised at an age of 14 days. Excision earlier or later resulted in less roots, less total root length, and shorter life-span.     

Effects of 6-Benzylaminopurine and Potassium on {delta}-Aminolevulinate Dehydratase Activity of Excised Cucumber Cotyledons

Changes in -aminolevulinate dehydratase (ALAD) activity werefollowed in 7-day excised cotyledons maintained on water ortreated with 6-benzylaminopurine (BA 1 µg.ml^–1),KCl (10 mM) or a combination of BA + KCl. Cotyledons were incubatedfor 3 days in the dark or in the light, or preincubated for18 h in the dark before illumination. Cycloheximide treatments(10µg.ml^–1) were performed to differenciate betweenALAD biosynthesis or activation. The excision entailed an increase in ALAD activity. Then a decreaseappeared after a 24-h incubation on water in the dark. It wasmore precocious in the light, but was delayed when the cotyledonswere illuminated after a dark preincubation. BA had little effect on ALAD activity in the dark. In the light,BA completely prevented the decrease in ALAD activity observedin the control, and enhanced this activity when a dark preincubationpreceded illumination. KCl had a slight stimulating effect inthe dark, but was uneffective in the light. The combinationBA + KCl was always devoid of stimulating effect. Stimulatingeffects of BA or KCl seemed to be due to an increase in ALADbiosynthesis. No treatment markedly affected ALAD exportationfrom cytoplasm to plastids. These results are discussed in relation with cotyledon greening. (Received November 25, 1986; Accepted February 5, 1987)     

Effects of Benzylaminopurine and Abscisic Acid on Distribution of rRNA in the Palisade Cells of Excised Cucurbita Pepo Cotyledons

rRNA was labelled with RNase-gold complexes applied to ultrathin Lowicryl sections of excised Cucurbita pepo L. (zucchini) cotyledons grown in darkness. Benzylaminopurine-caused stimulation of cotyledon growth decreases the density of ribosomes in palisade cells despite stimulating rRNA synthesis. Abscisic acid inhibits RNA synthesis and transport, but does not visibly affect the number of pre-existing ribosomes in retarded growth cells. The amount of rRNA in the cells of 24 h treated cotyledons is rather consequence of the growth rate than its leading factor.