Retardation and Reversal of Senescence in Bean Leaves by Benzyladenine and Decapitation

During the early stages of growth when the primary leaves ofbeans (Phaseolus vulgaris L., cv. Kinghorn) are expanding, thereis an increase in chlorophylls, carotenoids and protein levelsas well as superoxide dismutase (SOD), catalase and peroxidaseactivities. However, following the initiation of flowering onday (d) 21 there is a steady decrease in all of these parametersexcept levels of peroxidase activity, and by d 35 the primaryleaves are also showing pronounced morphological symptoms ofsenescence. Following the onset of senescence, primary leaveswere also leaking and showed increased levels of malondialdehyde(MDA). In addition, there was an increase in the lipid phasetransition temperature of a membrane fraction consisting predominantlyof chloroplast membranes, which reflected the formation of gelphase lipid. By d 56, the leaves had abscinded. A single applicationof benzyladenine (BA) to the primary leaves or decapitationof the shoot above the primary leaf on d 9 retarded all of thesesenescence symptoms, with decapitation being more effectivethan BA. On d 35 when the primary leaves were pale green-yellow, a singleapplication of BA to the leaves or decapitation of the shootabove them reversed the symptoms of senescence. Decapitationwas more effective than BA as a means of reversal, causing chlorophyll,carotenoid and protein levels to rise to values higher thanthose present in the control leaves. Decapitation also reversedthe senescence-related changes in membrane phase properties,decreased membrane leakiness and reduced lipid peroxidation. (Received August 22, 1983; Accepted January 26, 1984)     

Ribonuclease and Chlorophyllase Activities in Senescing Leaves

The activities of two enzymes, ribonuclease and chlorophyllase were investigated during the senescence of leaves. Ribonuclease activities were measured in primary leaves of Phaseolus vulgaris, and related to the levels of nucleic acid, protein and chlorophyll. Similarly, changes in chlorophyllase activity during senescence of leaves of Raphanus sativus were measured and related to chlorophyll. During senescence the levels of each enzyme as well as its respective substrate declined. Retardation of senescence, by excision of young tissue from intact plants or by treatment of detached leaves with cytokinins resulted in a maintainace of both the substrate and enzyme levels. It was concluded that high levels of ribonuclease and chlorophyllase activity are not linked directly with the degradation of RNA and chlorophyll during leaf senescence.     

Effect of benzyladenine on RNA and protein synthesis in intact bean leaves at various stages of ageing

Primary leaves of intact bean plants ( Phaseolus vulgaris L.) were treated with benzyladenine (BA) at different stages of ageing, BA promoted the synthesis of RNA, and soluble and insoluble proteins. The effects of BA stimulation differed depending on the age at which the leaf received the hormone treatment. In leaves attached to the plant, BA appeared to stimulate the rate of synthesis more than the rate of decomposition of RNA and protein, resulting in a net increase in RNA and protein. Both chloroplast and cytoplasmic ribosomes were still observed in intact yellowish green leaves. Polysomes in the cytoplasm increased remarkably when BA treatment was begun at late stages.     

Retardation of bean leaf senescence by benzyladenine and its influence on phosphate metabolism

The levels of glucose, sugar phosphates, and adenosine phosphates were determined in primary leaves of intact bean plants during normal senescence and compared to leaves in which senescence was delayed by application of benzyladenine (BA). In both cases there was a rise with time in the levels of glucose 1-phosphate, glucose 6-phosphate, and fructose 6-phosphate, and a decline in 2-phosphoglyceric acid, inorganic phosphate, and the adenosine phosphates (AMP, ADP, ATP). The levels of fructose 1,6-diphosphate remained fairly constant. Although the levels of hexose phosphates, adenosine phosphates, and inorganic phosphate were lower in the BA-treated leaves, the incorporation of ³²P into these compounds by 3- and 6-week-old plants was higher than in the controls. These results suggest that the retardation of leaf senescence by BA in intact bean plants is associated with increased utilization of metabolites, indicating a more rapid turnover of the adenosine phosphates. It is concluded that this effect is brought about by a regulatory coordination of metabolic processes in relation to energy production and utilization.     

The Effect of Benzyladenine on Changes in Nuclease and Protease Activities in Intact Bean Leaves during Ageing

Primary leaves of intact bean plants (Phaseolus vulgaris L.) were treated with benzyladenine (BA) at different stages during growth. Changes in DNase, RNase, and proteas activities in the leaves were followed. Unlike the case of various excised tissues, cytokinin raised the activities of these hydrolases in intact bean leaves. Because BA elevated the levels of DNA, RNA, and protein in intact leaves, it may stimulate both synthesis and decomposition of these cellular constituents. The hydrolase activities showed differential responses to BA according to the age at which the leaf received the hormone treatment.     

A gene encoding an acyl hydrolase is involved in leaf senescence in Arabidopsis

SAG101, a leaf senescence-associated gene, was cloned from an Arabidopsis leaf senescence enhancer trap line and functionally characterized. Reporter gene and RNA gel blot analyses revealed that SAG101 was not expressed until the onset of senescence in leaves. A recombinant SAG101 fusion protein overexpressed in Escherichia coli displayed acyl hydrolase activity. Antisense RNA interference in transgenic plants delayed the onset of leaf senescence for approximately 4 days. Chemically induced overexpression of SAG101 caused precocious senescence in both attached and detached leaves of transgenic Arabidopsis plants. These data suggest that SAG101 plays a significant role in leaf senescence.     

Effects of Benzyladenine on Chlorophyll, DNA, RNA and Protein Content of Attached Young Bean (Phaseolus vulgaris L.) Leaves

N⁶-Benzyladenine (BA) was applied to intact bean (Phaseolusvulgaris L.) primary leaves at 2 and 6 days after imbibition,when they were in the cell division and post-cell division stages,respectively. BA treatment at day 2 temporarily inhibited an increase in chlorophyllcontent in the following day, but stimulated it in later days.No such inhibition by BA was observed for changes with timein DNA, RNA, and protein content and f. wt. On the other hand,BA treatment at day 6 enhanced RNA and protein content, withoutsignificant influence on DNA and chlorophyll content and f.wt. The mode of cytokinin action on greening in leaves during cell-divisiongrowth seems to be different from that in etiolated cotyledons. Phaseolus vulgaris L., bean, greening, benzyladenine, DNA, RNA, protein     

Differential effects of benzyladenine on the ultrastructure of chloroplasts in intact bean leaves according to their age

Summary Primary leaves of intact bean plants (Phaseolus vulgaris) were treated with benzyladenine (BA) at different stages of growth. Changes in the ultrastructure of chloroplasts and the contents of chlorophyll, carotenoid, and protein (soluble and insoluble) in leaves with different treatments were followed and compared. When BA was applied from an early stage, it increased the chloroplast size and the number of grana per chloroplast without any pronounced effect on the grana size. When BA treatment was stopped at the early stage, these effects remained for a while and then diminished. When BA treatment was begun at a late stage, such marked effects were not observed, suggesting that only young leaves could respond to BA in that manner. However, the late treatment efficiently prevented the process of the last stage of leaf senescence characterized by disintegration of thylakoids with concomitant increase in the plastoglobule size. Chlorophyll, carotenoid, and insoluble protein contents per leaf followed similar changes in chloroplast length and the number of grana per chloroplast section.     

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.     

Ribonuclease in Leaves of Phaseolus vulgaris during Maturation and Senescence

The levels of the enzyme ribonuclease (RNase) were determined in primary leaves of Phaseolus vulgaris in an attempt to correlate changes in RNA with maturation and senescence. RNase, RNA and chlorophyll levels increased in expanding and maturing tissue and subsequently declined in senescing tissue. Senescence of the primary leaves started with the onset of flowering. A simultaneous increase of RNA and RNase in maturing tissue and a decrease during senescence suggests that the enzyme activity is correlated with the rate of‘turnover’of RNA rather than the absolute levels of RNA present in the tissue.     

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.     

Retardation of senescence by low temperature and benzyladenine in intact primary leaves of soybean

Effects of temperature and benzyladenine (BA) on the senescenceof intact primary leaves of soybean were investigated. Comparedwith high temperature (30?C for day and 25?C for night), lowtemperature (15?C for day and 13?C for night) significantlyretarded senescence of intact primary leaves. Repeated dailytreatment of the primary leaves with BA (200 mg/liter) beginning15 days after growth at high temperature resulted in retardationof the senescence process. The lower activity of cytokininsin the primary leaves of seedlings grown under high temperaturemay be responsible for rapid senescence. (Received January 14, 1980; )     

大豆叶片衰老过程中质膜蛋白激酶自磷酸化状态和催化活性的变化

以大豆幼苗初生叶为材料研究了衰老过程中质膜蛋白激酶自磷酸化状态和催化活性的变化。结果发现质膜上一个57kD的蛋白激酶分子上有多个自磷酸化位点,而且自磷酸化反应能提高该酶催化组蛋白H1磷酸化的激酶活力。进一步的研究表明诱导衰老处理造成的57kD蛋白激酶自磷酸化状态的变化,可能对调节它在衰老过程中催化活性的变化起重要作用;而外源6-BA预处理则能够维持57kD蛋白激酶体内高自磷酸化状态,保持该激酶在衰老过程中的催化活力。对衰老和6-BA处理过程中质膜上39和47kD蛋白激酶自磷酸化状态变化的研究表明,这两种激酶可能参与大豆叶片对6-BA刺激信号的传导和/或应答反应过程。     

Effects of light and regulators on senescence-related changes in soluble proteins in detached oat (Avena sativa L.) leaves

The rate of senescence and the two-dimensional pattern of soluble proteins from detached oat leaves senescing in either darkness or light were analyzed, and compared to those of leaves in which senescence was delayed by application of the cytokinin benzyladenine or enhanced through the action of abscisic acid.Senescence of detached leaves in light did not differ significantly from senescence in attached leaves on intact plants. In darkness, protein was lost at a higher rate than in light, but several individual proteins showed relative increases. Notably, proteins previously characterized as high-molecular-weight proteins and senescence-associated proteins (Klerk et al., 1992) increased. Changes observed during incubation in light or darkness appeared to be related to this condition rather than the rate or progress of senescence. Cytokinins delayed and abscisic acid accelerated the changes in protein pattern compared to water. Beside changes previously identified in leaves senescing on the plant, detached leaves show alterations that reflect their condition of incubation rather than their developmental progress.Abbreviations 2D-PAG two-dimensional polyacrylamide gel electrophoresis - ABA abscisic acid - BA N⁶-benzyladenine - BSA bovine serum albumin - EDTA ethylenediamine tetraacetic acid - IEF isoelectric focusing - Rubisco ribulosebisphosphate carboxylase/oxygenase - SDS sodium dodecyl sulfate - Tris tris (hydroxymethyl) aminomethane