Effect of Benzyladenine on DNA, RNA, Protein, and Chlorophyll Contents in Intact Bean Leaves: Differential Responses to Benzyladenine According to Leaf Age

Benzyladenine (BA) was applied to intact bean (Phaseolus vulgaris) leaves at different stages of their growth. Changes in the amounts of cellular constituents resulting from the different treatments were followed and compared. RNA, protein, and chlorophyll contents, dry weight, fresh weight, and leaf area per single leaf continued increasing when leaves were treated with BA from an early stage, whereas in untreated leaves all these values levelled off or declined with advancing age. Besides these changes, BA treatment induced an increase in the DNA content. Changes in RNA content was more remarkable in response to application or deprival of BA treatment than the corresponding ones in protein and chlorophyll contents. The pattern of response to BA varied greatly according to the age at which the leaf received the treatment. As leaves aged, they lost the ability to increase their area and fresh weight in response to BA. However, continuous treatment with BA from an early stage kept the leaves young and able to respond.     

Effects of Benzyladenine on Bean Leaf Senescence and the Translocation of 14C-Assimilates

Treating primary leaves of bean plants with benzyladenine (BA) greatly increased the retention of photosynthetic assimilates in the treated leaves. Within 24 hours of treatment, the BA treated primary leaves retained 70 % of their assimilates and maintained this high level throughout the period studied. In contrast, the primary leaves of control plants retained 30 % at week 2, increased retention to 80 % between week 4 and 5 and dropped to 50 % during senescence at week 6. When the trifoliate leaves of 5 week old plants were fed ¹⁴CO₂, less than 1 % of the total activity was recovered from the BA treated leaves. It is concluded that the retardation of leaf senescence by BA on intact plants is not due to mobilization of metabolites from other plant parts, but is associated with a high retention of photosynthates.     

Benzyladenine-induced increase in DNA content per chloroplast in intact bean leaves

Benzyladenine (BA) treatment was found to induce chloroplast DNA (ctDNA) synthesis after it had stopped in primary leaves of light-grown intact bean plants (Phaseolus vulgaris L.). The leaves were treated with BA from 7 days after sowing. Chloroplasts were isolated and the ctDNA content per chloroplast was determined. Chloroplast division occurred until 13 days after sowing in untreated leaves. BA stimulated the division keeping the level of ctDNA content per chloroplast the same as that in the untreated controls. After the division period, the ctDNA content per chloroplast increased in BA-treated leaves, but not in controls. Consequently, ctDNA per leaf (or per cell) increased immediately after the beginning of BA treatment, but remained constant in the control leaves.     

Effect of Benzyladenine on Diurnal Changes in DNA Content per Chloroplast and Chloroplast Replication in Intact Bean Leaves

The effect of benzyladenine (BA) on the diurnal changes in DNAand Chl contents per chloroplast and chloroplast replicationin primary leaves of bean plants (Phaseolus vulgaris L.) grownunder a 16 h light/8 h dark cycle was studied. Experiments weremade on primary leaves in the early expansion phase, where celldivision had been completed but chloroplasts were replicating.In untreated controls, chloroplast number, Chl content and freshweight per leaf showed daily periodic changes. Chl content perchloroplast increased in the light period every day, and freshweight per leaf increased most rapidly in the early dark period.Chloroplast number per leaf increased rapidly in the early darkperiod on day 9, though the increase began a little earlierand was less sharp on days 8 and 10. During these periods, DNAcontent per chloroplast was decreasing due to chloroplast divisionas chloroplast DNA (ctDNA) per leaf remained unchanged throughoutthe experimental period. BA induced increases in Chi contentper chloroplast, ctDNA content and fresh weight per leaf within6 h of its application, regardless of whether it was appliedat or 10 h after the beginning of the light period. Applicationof BA at 10 h in the light period shifted the start of chloroplastreplication by 6 h compared to that in untreated controls. However,when BA was applied at the beginning of illumination, the startof chloroplast replication showed the same relative change intime as above. 5-Fluorodeoxyuridine (5-FdU) promptly preventedBA-induced increase in Chl content and chloroplast number perleaf as well as ctDNA content per leaf.     

An explanation for the light requirement of AgNO3 to inhibit ethylene-induced abscission

The loss of the antiethylene activity of Ag⁺ on leaf abscission by incubation in the dark was investigated. When primary leaves were removed from cuttings of Vigna radiata previously sprayed with AgNO₃, dark-induced abscission of the petioles was inhibited, compared to untreated leafless controls, in the presence or absence of ethephon, an ethylene-releasing compound. Malformin did not negate inhibition of petiole abscission induced by Ag⁺. Although leaf removal restored the antiethylene activity of Ag⁺ in the dark, macerates of leaves from dark-aged cuttings did not negate the ability of Ag⁺ to inhibit petiole abscission in the dark. Abscisic acid completely abolished the ability of Ag⁺ to counteract ethephon-induced leaf abscission in the light, and almost completely abolished the Ag⁺-induced inhibition of petiole abscission from explants in the dark. It is proposed that the phytochrome requirement for the antiethylene activity of Ag⁺ on ethephon-induced leaf abscission involves prevention of the formation, accumulation, or transport of a substance in leaves in the dark which negates Ag⁺ activity. This substance may be abscisic acid or another substance with similar biological activity.     

Effects of Benzyladenine on Photosynthesis, Growth and Senescence of the Bean Plant

The net photosynthetic rate of attached primary bean leaves treated with benzyladenine (BA) decreased from 35 nano-grams carbon dioxide per square centimetre and second (ng cm^?2 s^?1) at week 2 to 17 ng cm^?2 s^?1 at week 4, and thereafter increased to 24 at week 6. By contrast the net photo-synthetic rate of the water-treated leaves decreased to 4–5 ng cm^?2 s^?1 at weeks 5 and 6. The stomatal resistance was not markedly affected by BA treatment. The BA-treated primary leaves had higher dry and specific weights, and the chlorophyll and carotenoid contents were greater than for the water controls. The pigment content of the BA-treated leaves steadily increased from week 2 to 6, whereas in the water controls it remained constant till week 4 and thereafter decreased. It is concluded that retardation of leaf senescence by BA is assoclatcd with a mnintenance of photosynthetic activity.     

Sequence of morphological and physiological events during natural ageing and senescence of a castor bean leaf: sieve tube occlusion and carbohydrate back-up precede chlorophyll degradation

The development of castor bean ( Ricinus communis L. var. sanguineus) leaves from bud break to abscission was studied to determine whether senescence of phloem precedes or follows chlorophyll degradation in the course of natural ageing of leaves. The castor bean leaf blade took 20 days for full expansion and its average life span was 60 days. From the day of full expansion on it suffered a substantial loss in N, a small loss in C, K and P and a gain in Ca, Mg and S. The content of soluble sugars increased with time, paralleled by a decrease of photosynthetic activity. Starch accumulated shortly before chlorophyll breakdown. The amino acid level in the leaves decreased steadily together with nitrate reductase and glutamine synthetase activity. Reactive oxygen species increased and oxidation-protecting compounds decreased during the life span of the leaves. Shortly after full leaf expansion an increasing number of sieve plates showed strong callose depositions when visualized by aniline blue method. At day 40 only half of the sieve tubes appeared functional. Chlorophyll breakdown followed these processes with a time lag of approximately 10 days. The sieve tube sap of ageing leaves had the same sucrose concentrations as young leaves, whereas amino acid concentrations decreased. High levels of reduced ascorbic acid and glutathione together with increasing levels of glutaredoxin indicated oxidative strain during senescence. We speculate that the gradual increase of reactive oxygen species during ageing together with the import of calcium ions lead to the stimulation of callose synthesis in plasmodesmata and sieve plates with the consequence of inhibition of phloem transport leading to carbohydrate back-up in the leaf blade. The latter may finally induce chlorophyll breakdown and, at the end, leaf abscission at the petiole base. Thus phloem blockage would precede and may be causal for chlorophyll degradation in leaf senescence.     

Effects of Age on Sodium Fluxes in Primary Bean Leaves

The effects of age on sodium fluxes in primary-leaf sections from control and decapitated bean plants were determined. It was assumed that at high external salt concentrations the measurements represent mainly fluxes through the tonoplast, and that in brief efflux experiment subsequent to free-space exchange efflux from the cytoplasma is essentially measured. Net- and influxes to the vacuoles increase during the period of leaf expansion and net chlorophyll synthesis. Both fluxes and amount of chlorophyll decrease rapidly in senescing leaves. The exchangeability of previously absorbed sodium also increased in senescing leaves. Decapitation delayed senescence and very much reduced the decreases in net flux and influx, and the exchangeability of sodium. The results are discussed in relation to solutes redistribution in the plant.     

Relative rates of delivery of xylem solute to shoot tissues: Possible relationship to sequential leaf senescence

The rates of delivery of regulatory solutes such as cytokinins and mineral ions from the roots to competing shoot tissues can influence rates of metabolism and development. A 15 min pulse of a synthetic xylem mobile and phloem-immobile solute, acid fuchsin, was used to quantify relative rates of solute delivery to competing organs on excised transpiring bean shoots (Phaseolus vulgaris L. cv. Contender) at different stages of development. Stem, flower and fruit tissues received comparatively low rates of solute delivery. The relative rate of solute delivery to newly opened leaves was initially low, but increased during rapid leaf expansion and then declined progressively as the leaves exceeded 70% of their final area. The relative rate of solute delivery to tissues of the basal primary leaves declined progressively from 2 weeks onwards. This decline appeared to be caused by progressive internally regulated increases in both stomatal resistances and hydraulic resistances to xylem flow up to and into the leaf blade. Thus combined abaxial and adaxial stomatal resistance values in the primary leaves (Rs) increased from 3 to ≥ 7 s cm^?1 between 2 and 5 weeks. Similarly, mean values for the connection resistances (Rc) to hydraulic flow into the primary leaves rose from 7 to 13 TPa · s · m^?1 between 2 and 4 weeks. In the same period pathway resistance from stem to primary leaf petioles (Rp), as determined by direct pressure flow assay, increased from 7 to 15 TPa · s · m^?1. Senescence-associated declines in protein and chlorophyll levels in the primary leaves were initiated in parallel with, or after, declines in relative rates of solute delivery. The provision of extra illumination at the basal leaf level between 2 and 5 weeks did not prevent declines in chlorophyll and soluble protein or increases in stomatal resistance. We suggest that internally programmed changes in the hydraulic architecture of the plant progressively divert xylem-transported root supplies of nutrients and cytokinins from basal to more apical leaves and thus regulate the progressive senescence of leaves from the base upwards.     

Involvement of ethylene in the action of the cotton defoliant thidiazuron

The effect of the defoliant thidiazuron (N-phenyl-N′-1,2,3-thiadiazol-5-ylurea) on endogenous ethylene evolution and the role of endogenous ethylene in thidiazuron-mediated leaf abscission were examined in cotton (Gossypium hirsutum L. cv Stoneville 519) seedlings. Treatment of 20- to 30-day-old seedlings with thidiazuron at concentrations equal to or greater than 10 micromolar resulted in leaf abscission. At a treatment concentration of 100 micromolar, nearly total abscission of the youngest leaves was observed. Following treatment, abscission of the younger leaves commenced within 48 hours and was complete by 120 hours. A large increase in ethylene evolution from leaf blades and abscission zone explants was readily detectable within 24 hours of treatment and persisted until leaf fall. Ethylene evolution from treated leaf blades was greatest 1 day posttreatment and reached levels in excess of 600 nanoliters per gram fresh weight per hour (26.7 nanomoles per gram fresh weight per hour). The increase in ethylene evolution occurred in the absence of increased ethane evolution, altered leaf water potential, or decreased chlorophyll levels. Treatment of seedlings with inhibitors of ethylene action (silver thiosulfate, hypobaric pressure) or ethylene synthesis (aminoethoxyvinylglycine) resulted in an inhibition of thidiazuron-induced defoliation. Application of exogenous ethylene or 1-aminocyclopropane-1-carboxylic acid largely restored the thidiazuron response. The results indicate that thidiazuron-induced leaf abscission is mediated, at least in part, by an increase in endogenous ethylene evolution. However, alterations of other phytohormone systems thought to be involved in regulating leaf abscission are not excluded by these studies.     

Effects of salt on growth,ion accumulation,photosynthesis and leaf anatomy of the mangrove,<Emphasis Type="Italic"> Bruguiera parviflora</Emphasis>

The effects of a range of salinity (0, 100, 200 and 400 mM NaCl) on growth, ion accumulation, photosynthesis and anatomical changes of leaves were studied in the mangrove, Bruguiera parviflora of the family Rhizophoraceae under hydroponically cultured conditions. The growth rates measured in terms of plant height, fresh and dry weight and leaf area were maximal in culture treated with 100 mM NaCl and decreased at higher concentrations. A significant increase of Na⁺ content of leaves from 46.01 mmol m^-2 in the absence of NaCl to 140.55 mmol m^-2 in plants treated with 400 mM NaCl was recorded. The corresponding Cl^- contents were 26.92 mmol m^-2 and 97.89 mmol m^-2. There was no significant alteration of the endogenous level of K⁺ and Fe²⁺ in leaves. A drop of Ca²⁺ and Mg²⁺ content of leaves upon salt accumulation suggests increasing membrane stability and decreased chlorophyll content respectively. Total chlorophyll content decreased from 83.44 g cm^-2 in untreated plants to 46.56 g cm^-2 in plants treated with 400 mM NaCl, suggesting that NaCl has a limiting effect on photochemistry that ultimately affects photosynthesis by inhibiting chlorophyll synthesis (ca. 50% loss in chlorophyll). Light-saturated rates of photosynthesis decreased by 22% in plants treated with 400 mM NaCl compared with untreated plants. Both mesophyll and stomatal conductance by CO₂ diffusion decreased linearly in leaves with increasing salt concentration. Stomatal and mesophyll conductance decreased by 49% and 52% respectively after 45 days in 400 mM NaCl compared with conductance in the absence of NaCl. Scanning electron microscope study revealed a decreased stomatal pore area (63%) in plants treated with 400 mM NaCl compared with untreated plants, which might be responsible for decreased stomatal conductance. Epidermal and mesophyll thickness and intercellular spaces decreased significantly in leaves after treatment with 400 mM NaCl compared with untreated leaves. These changes in mesophyll anatomy might have accounted for the decreased mesophyll conductance. We conclude that high salinity reduces photosynthesis in leaves of B. parviflora, primarily by reducing diffusion of CO₂ to the chloroplast, both by stomatal closure and by changes in mesophyll structure, which decreased the conductance to CO₂ within the leaf, as well as by affecting the photochemistry of the leaves.     

Quantification of the kinetin effect on protein synthesis and degradation in senescing wheat leaves

Wheat leaves (Triticum aestivum L. cv San Agustin INTA) were detached when they reached maximum expansion, put individually in tubes containing water and left in darkness. After 3 days the protein content had decreased to 46% of the initial value. When the leaves were placed in 1 micromolar kinetin, they retained 60% of the initial protein content for the same period. This effect was observed only when leaves were treated with kinetin within the first 24 hours after detachment. The action of kinetin on both protein synthesis and degradation was quantitatively measured. Synthesis was estimated by the incorporation of l-[³H]leucine into proteins. It was higher in kinetin treated than in non treated leaves. It contributed to about 14 micrograms of protein retention per leaf in 3 days. Measurement of protein degradation, evaluated by the decay of radioactivity in leaf proteins previously labeled with l-[³H] leucine or as the difference between rates of protein synthesis and protein content, showed that kinetin decreased protein breakdown rates. It accounted for about 186 micrograms of protein retention per leaf in 3 days. Hence, kinetin action on protein breakdown was 13-fold average higher than its action on synthesis for the conservation of leaf protein. This difference is higher in early stages of the process.     

Time course of antioxidant responses of Capsicum annuum subjected to a progressive magnesium deficiency

The effect of magnesium (Mg²⁺)‐deficiency on the antioxidant responses of Capsicum annuum was investigated over a 60‐day period under controlled conditions. This Mg²⁺‐deficiency aimed to mimic the physiological conditions that plants may experience in the field. At each harvest time, five different leaf‐levels (L2 to L6) were distinguished. L2 and L6 correspond to the second and sixth youngest leaves, respectively. The following parameters were determined: Mg²⁺, chlorophyll and protein contents, total and redox pools of ascorbate and glutathione, and the activities of superoxide dismutase, ascorbate peroxidase, dehydroascorbate reductase, and glutathione reductase. Under Mg²⁺‐deficiency, leaf Mg²⁺ contents decreased over time in all leaf‐levels except in the second youngest leaves (L2), where they remained constant at about 0.25% (dry weight basis). Mg²⁺‐deficiency led to an increase in the antioxidant enzyme activities concomitant with an increase in the ascorbate and glutathione pools, whereas total chlorophyll and soluble protein contents decreased. The L2 leaves showed an increase in glutathione reductase activity and in the ascorbate redox state whereas no difference was observed for the other parameters. Superoxide dismutase activities increased in L5 leaves from day 15 and, afterwards, in L3 to L5 leaves, irrespective of Mg²⁺ content. At day 30, glutathione reductase activities increased in L2 to L4 leaves and dehydroascorbate reductase activities in L4 leaves. At day 45, we observed an increase in the ascorbate peroxidase activities in L3 to L5 leaves. At the same time, ascorbate and glutathione pools increased in intermediate leaves, whereas chlorophyll content decreased in L3 and L4 leaves, and protein content decreased in L4 leaves. Results suggest that pepper leaves enhance their defence capacities against oxidative stress by increasing ascorbate more than glutathione synthesis. However, cells showed higher regeneration rates for the glutathione redox state than for the ascorbate redox state.     

Effect of exposure to subfreezing temperatures on ethylene evolution and leaf abscission in citrus

Citrus leaves exposed to subfreezing temperatures evolved ethylene at rates between 0.1 and 38.3 microliters per kilogram fresh weight per hour whereas untreated leaves evolved between 0.01 and 0.50 microliter per kilogram fresh weight per hour. Leaves not injured by freezing temperatures did not abscise, and ethylene evolution was near normal after 2 days. Freeze-injured leaves continued evolving high ethylene levels 4 or 5 days subsequent to freeze injury, and many of the freeze-killed leaves abscised. Supportive evidence suggested freeze-induced ethylene was involved in freeze-induced leaf abscission; whereas freeze-inhibited abscission was not due to a lack of ethylene but injury to other metabolic systems necessary for abscission.     

Influence of Benzyladenine, Leaf Darkening, and Ringing on Movement of C-labeled Assimilates Into Expanded Leaves of Vitis vinifera L

Leaves of Vitis vinifera L., nearly fully expanded, imported only trace amounts of ¹⁴C following assimilation of ¹⁴CO₂ by a lower leaf on the same shoot, but benzyladenine (BA) application at 4.4 × 10⁻³m caused a marked increase in the movement of ¹⁴C into these leaves. Older leaves near the shoot base were less responsive; BA treatment alone had little effect on import of labeled assimilates from adjacent leaves but when the BA-treated leaves were darkened there was an increased import of labeled materials. When these 2 treatments were combined and applied to leaves on shoots with ringed bases, relatively high levels of radioactivity were detected in the BA-treated leaves but under these conditions darkening, without the application of BA, also resulted in an increased import of ¹⁴C. Accumulation of imported ¹⁴C was found to be restricted to the area of the leaf blade treated with BA. Separation of labeled compounds in ethanol extracts of treated leaves showed a lower percentage of radioactivity present in the sugar fraction from BA-treated leaves and an increased percentage present in the amino acid fraction.     

Influence of Different Cytokinins on the Transpiration and Senescence of Excised Oat Leaves

In order to investigate the possibility that cytokinins control transpiration indirectly through affecting leaf senescence, a direct comparison was made of the effect of different cytokinins on transpiration and senescence of oat leaves (Avena sativa L. cv. Forward). Senescence was assessed by measuring chlorophyll loss. The synthetic cytokinins N⁶ benzyladenine (BA) and kinetin delayed senescence and increased transpiration of oat leaves to a greater extent than did the naturally occurring compounds zeatin, N^b-Δ² isopentenyladenine (i⁶ Ade) and 6-ø-hydroxybenzyladenosine (hyd-BA riboside). During the early stages of the transpiration experiment zeatin showed similar or greater activity than BA. This period was longest when freshly excised leaves were used, was reduced when leaves were used after incubation in distilled water in the dark for 20 h and was eliminated by incubation in cytokinin solution in the dark. After this period the activity of zeatin declined relative to BA. The effect of cytokinins in increasing transpiration occurred only in the light; no effect was observed in the dark. BA showed higher activity than zeatin in senescence tests but both cytokinins were less effective as the tests progressed, this decrease in activity being more rapid when older leaves were used. The results are discussed in relation to the mechanisms by which endogenous cytokinins might control sensecence and transpiration in oat leaves and to the value of the oat leaf senscence and transpiration bioassays as tests for cytokinin activity of plant extracts.     

Functional and Structural Changes in Senescing Populus deltoides (Bartr.) Chloroplasts

Chloroplasts isolated from Populus deltoides leaves were used to study age-dependent changes in the rate of cyclic photophosphorylation. Single leaves were used to measure CO₂ fixation by leaf discs, chlorophyll concentration, and ATP synthesis. The ability of chloroplasts to synthesize ATP diminished steadily from the time of full leaf expansion, regardless whether the results are expressed on a leaf area or chlorophyll basis. This decline in the rates of ATP synthesis was paralleled by the decline in the rate of CO₂ fixation. The results suggest that the efficiency of the membrane-bound ATP synthesizing system declines with age.     

Patterns of ehtylene production in senescing leaves

Changes in the patterns of ethylene production, chlorophyll content, and respiration were studied in relation to the senescence of intact leaves and leaf discs. The primary leaves of pinto bean, which abscise readily during natural senescence, and tobacco and sugar beet leaves, which do not abscise, were used. A decrease in the rate of ethylene production and respiration, during the slow phase of chlorophyll degradation, was observed in leaf-blade discs cut from mature leaves and aged in the dark. During rapid chlorophyll loss both ethylene production and respiration increased and then decreased. These climacteric-like patterns were shown by leaf discs of all three species. Discs taken from leaves that had been senescing on the plant also showed a climacteric-like rise in ethylene production but not in respiration, which decreased continuously with leaf age. Climacteric-like patterns in the rise of ethylene and respiration for leaf discs were also shown by the petioles of both bean and tobacco leaves. This indicates that the rise of ethylene and respiration is characteristic of the general process of senescence in leaves and is not restricted to the abscission process. In contrast to the ethylene-forming systems in climacteric fruits and many flowers, the one in leaves declines sharply in the early stages of senescence. The subsequent rise of ethylene production appears to be associated with the rapid phase of chlorophyll breakdown, and may indicate the final stage of the senescence process during which ethylene could be actively involved in inducing leaf abscission.     

甘蔗成熟期叶鞘纤维素酶活性对自然脱落的影响

采用脱叶性不同的4个甘蔗品种为材料,研究了在成熟期叶鞘纤维素酶活性对蔗叶脱落的影响。结果表明,纤维素酶活性影响甘蔗叶片脱落,在收获期叶鞘纤维素酶活性与植株脱落率均达到最高。不同叶位中,＋10叶位纤维素酶活性最高。相关性分析表明,4个甘蔗品种的＋10叶位叶鞘纤维素酶活性与脱落率呈正相关。