Breakdown of Ribulose Bisphosphate Carboxylase and Change in Proteolytic Activity during Dark-induced Senescence of Wheat Seedlings

When 8-day-old wheat seedlings (Triticum aestivum L. var. Chris) are placed in the dark the fully expanded primary leaves undergo the normal changes associated with senescence, for example, loss of chlorophyll, soluble protein, and photosynthetic capacity (Wittenbach 1977 Plant Physiol. 59: 1039-1042). Senescence in this leaf is completely reversible when plants are transferred to the light during the first 2 days, but thereafter it becomes an irreversible process. During the reversible stage of senescence the loss of ribulose bisphosphate carboxylase (RuBPCase) quantitated immunochemically, accounted for 80% of the total loss of soluble protein. There was no significant change in RuBPCase activity per milligram of antibody-recognized carboxylase during this stage despite an apparent decline in specific activity on a milligram of soluble protein basis. With the onset of the irreversible stage of senescence there was a rapid decline in activity per milligram of carboxylase, suggesting a loss of active sites. There was no increase in total proteolytic activity during the reversible stage of senescence despite the loss of carboxylase, indicating that this initial loss was not due to an increase in total activity. An 80% increase in proteolytic activity was correlated with the onset of the irreversible stage and the rapid decline in RuBPCase activity per milligram of carboxylase. Delaying senescence with zeatin reduced the rate of loss of carboxylase and delayed both the onset of the irreversible stage and the increase in proteolytic activity to the same degree, suggesting that these events are closely related. The main proteinases present in wheat and responsible for the increase in activity are the thiol proteinases. These proteinases have a high affinity for RuBPCase, exhibiting an apparent K_m at 38 C of 1.8 × 10⁻⁷ m. The K_m for casein was 1.1 × 10⁻⁶ m. If casein is representative of noncarboxylase protein, then the higher affinity for carboxylase may provide an explanation for its apparent preferential loss during the reversible stage of senescence.     

Reversible dark-induced senescence of soybean root nodules

Nodule senescence was induced in intact soybean [Glycine max. (L.) Merr., cv Woodworth] plants by an 8-day dark treatment. Dark-induced senescence resulted in the complete loss of acetylene reduction activity, a 67% loss of total soluble protein, and an almost complete loss in total leghemoglobin of nodule extracts. Isoelectric focusing gels demonstrated a preferential loss of certain proteins, which was correlated with an increase in endoprotease specific activity toward azocasein. Nodules were completely green after the 8-day dark treatment. If plants were returned to a normal photoperiod after 8 days in the dark, nodules recovered from the dark treatment in 12 to 16 days. Acetylene reduction activity returned to normal, and both total soluble protein and leghemoglobin were resynthesized while protease activity against azocasein decreased to the level of control nodules. The nodule population that had turned green after 8 days in the dark exhibited a progressive increase in red color starting nearest the exterior of the nodule, and after 16 days of recovery nodules were indistinguishable from control nodules maintained under a normal photoperiod.     

Effect of pod removal on leaf photosynthesis and soluble protein composition of field-grown soybeans

Well nodulated, field-grown soybeans (Glycine max [L.] Merr. var Williams) were depodded just prior to seed development and near mid pod-fill. Both treatments caused a considerable increase in leaf dry weight, suggesting continued photosynthate production following pod removal. Moreover, depodding had a marked effect on leaf soluble protein without affecting total proteolytic activity. Early depodding caused a 50% increase in leaf protein, and both early and late depodding caused the retention of protein for several weeks following the decline in control leaves. But despite this retention of protein, leaves of depodded plants showed no difference in the onset of the irreversible decline in photosynthesis. Therefore, although depodding delayed the loss of leaf chlorophyll and protein, it did not delay the onset of functional leaf senescence and in fact, actually appeared to enhance the rate of decline in photosynthesis. There was a good correlation between the irreversible decline in ribulose bisphosphate carboxylase (activity and amount) and that of photosynthesis. In contrast, the correlation did not seem as good between stomatal closure and the onset of the irreversible decline in photosynthesis. The reason total soluble protein remained high following depodding while carboxylase, which normally comprised 40% of the soluble protein, declined was because several polypeptides increased in amounts sufficient to offset the loss of carboxylase. This change in leaf protein composition indicates a change in leaf function; this is discussed in terms of other recent findings.     

Changes in endogenous cytokinin levels in cotyledons of Cucurbita pepo (zucchini) during natural and dark-induced senescence

Cytokinin (CK) levels in cotyledons of Cucurbita pepo L. (zucchini) were investigated through the processes of post-germination, greening, natural senescence and subsequent rejuvenation. The concentrations of the physiologically active CK bases, ribosides and nucleotides, as well as the cis -isomers of zeatin derivatives, decreased between the first and fifth weeks of cultivation under controlled light conditions. At the same time, the levels of storage CK O -glucosides and physiologically inactive CK 7- and 9-glucosides increased with senescence. With plant decapitation and subsequent cotyledon rejuvenation, not only the chlorophyll content but also the levels of physiologically active CKs, nucleotides and cis -zeatin derivatives increased. The levels of O -glucosides, however, decreased. When 1-week-old seedlings were transferred to the dark, there was a progressive reduction in cotyledon chlorophyll content, deterioration of chloroplast ultrastructure and a decrease in physiologically active CKs and their nucleotides. In contrast with natural senescence, the storage CK O -glucosides decreased under dark conditions, suggesting different metabolic regulation of endogenous CK levels during natural and dark-induced senescence of zucchini cotyledons. The chlorophyll loss of dark-treated cotyledons could be partially reversed, even after 5 days, with return to light conditions. During this recovery, physiologically active CKs and their nucleotides again increased, whereas the storage CK O -glucosides and cis -zeatins decreased. The present results suggest that dark-induced destruction and subsequent restoration of chloroplasts during light shifts are controlled by changes in the levels of physiologically active CKs and their nucleotides.     

Expression of photosynthesis- and senescence-related genes during leaf development and senescence in silver birch (Betula pendula) seedlings

Leaf development and senescence were studied in greenhouse-grown silver birch ( Betula pendula Roth) seedlings over a period of 7 weeks. Prior to the experiment, leaves from 100 seedlings were marked for five sampling dates. Timing of the developmental phases was studied with biochemical analyses of total soluble protein, Rubisco protein, chlorophyll concentration and at the level of gene expression related to photosynthesis, energy metabolism, ethylene synthesis and protein degradation. During the sampling period, an initial increase in photosynthetic capacity could be seen, when expression of the Rubisco small subunit gene ( RbcS ) and Rubisco protein (EC 4.1.1.39) were examined. Down-regulation of photosynthesis, visible as a decrease in Rubisco protein and RbcS mRNA, started soon after full expansion of the leaves and processes related to senescence followed. mRNA accumulation for the ethylene-forming enzyme 1-aminocyclopropane-1-carboxylic acid oxidase increased first during the onset of senescence. Protein degradation was observed as a loss of soluble proteins, with a simultaneous increase in the leucine aminopeptidase (EC 3.4.11.1) mRNA levels. The mRNA levels of ribonuclease-like pathogenesis-related protein 10 also increased clearly during senescence, but the mitochondrial phosphate translocator mRNA showed only a slight increase. Chlorophyll concentration of the leaves decreased after the mRNA levels of these senescence-related genes had become more abundant.     

Relation between Respiration and Senescence in Oat Leaves

The respiration of excised oat (Avena sativa cv Victory) leaves and their sensitivity to inhibitors was followed during senescence under varied conditions. The respiration rate, which in controls reaches its peak on the third day in darkness, is lowered at the time of fastest loss of chlorophyll (as reported earlier) by seven unrelated reagents that all delay dark senescence. When senescence is delayed by white light or by cytokinins, the respiratory rise is correspondingly delayed. Kinetin and l-serine, which act as antagonists on senescence, also act as antagonists on the respiratory rate. However, an exception to this close correspondence between senescence and the respiratory rise is offered by the lower aliphatic alcohols, which delay dark senescence and yet accelerate the onset of the respiratory rise.     

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.     

Recovery of loss in chlorophyll and 2,6-dichlorophenol indophenol Hill reaction of isolated chloroplasts during dark induced aging of intact maize seedlings

Loss in the content of pigments and decline in the efficiency of thylakoid membranes to reduce 2,6-dichlorophenol indophenol (DCPIP) have been investigated during dark induced senescence of attached leaves of maize seedlings. The chlorophyll degradation during senescence is differentially inhibited by indoleacetic acid (IAA), gibberellic acid (GA) and kinetin. IAA and GA behave as mild senescence inhibitors in comparison to kinetin. However, in comparison to light, kinetin is relatively less efficient in counteracting senescence. Dark-induced loss in chlorophyll content is fully recovered by light when the dark incubation period is relatively short. The pattern of light recovery of loss in photoelectron transport during dark-aging is similar to the recovery kinetics of chlorophyll. Continuous kinetin treatment of dark-incubated seedlings inhibits the chlorophyll degradation but with decreased duration of kinetin treatment, the efficiency of the hormone to inhibit chlorophyll loss is reduced. The kinetin-induced inhibition of pigment loss is small in comparison with the effect of light.     

ACC synthase expression regulates leaf performance and drought tolerance in maize

Ethylene regulates entry into several types of plant developmental cell death and senescence programs besides mediating plant responses to biotic and abiotic stress. The response of cereals to conditions of drought includes loss of leaf function and premature onset of senescence in older leaves. In this study, ACC synthase ( ACS ) mutants, affecting the first step in ethylene biosynthesis, were isolated in maize and their effect on leaf function examined. Loss of ZmACS6 expression resulted in delayed leaf senescence under normal growth conditions and inhibited drought-induced senescence. Zmacs6 leaves continued to be photosynthetically active under both conditions indicating that leaf function was maintained. The delayed senescence phenotype associated with loss of ZmACS6 expression was complemented by exogenous ACC. Surprisingly, elevated levels of foliar chlorophyll, Rubisco, and soluble protein as well as improved leaf performance was observed for all Zmasc6 leaves, including young and fully expanded leaves which were far from initiating senescence. These observations suggest that ethylene may serve to regulate leaf performance throughout its lifespan as well as to determine the onset of natural senescence and mediate drought-induced senescence.     

Nucleic Acid and protein metabolism of senescing and regenerating soybean cotyledons

An alternative to the leaf disk system for studies of the metabolism of senescence is described. The progress of senescence of soybean (Glycine max L.) cotyledons is arrested when the epicotyl is removed. Epicotyl removal at 16 or 17 days reversed the decline in nucleic acid, protein, and chlorophyll content in the cotyledon. Epicotyl removal at 18 days did not reverse the decline in the above components, and therefore the progress of cotyledon has passed the point of no return. Cotyledons lost 90% of their nucleic acid and 80% of their protein before senescence became irreversible. The rate of recovery in various macromolecular components after epicotyl removal did not occur in an equal manner. Nucleic acid was regenerated at a faster rate than chlorophyll, which was regenerated at a faster rate than soluble protein. The heavy nucleic acid components (ribosomal and heavy ribosomal messenger fractions) regenerated at greater rates than did the soluble RNA or DNA. No label from ¹⁴CO₂ was incorporated into DNA of the cotyledons when the epicotyl was present but label was incorporated into DNA after epicotyl removal.     

Interaction between Senescence and Wounding in Oat Leaves

A study was made of the influence of wounding on the senescence of standard oat leaf segments in the dark. Wounding was by either subdividing the 3 centimeter long segments into 5 millimeter subsegments, gently scraping the adaxial surface of the segments with a sharp blade, making transverse linear cuts, or by making many small holes with a needle. Wounding considerably delayed the loss of both chlorophyll and protein in the dark and the amount of inhibition was roughly proportional to the intensity of wounding. With surface wounding, the inhibition of senescence was detectable from the first day of dark incubation; other methods caused moderate promotion of senescence for the first 2 days but decreased the loss of chlorophyll and protein thereafter. A number of senescence-modifying substances acted similarly on both unwounded and wounded segments, but the amount of chlorophyll and protein in the wounded segments was always more than in the respective controls. Cytokinins, however, provided an exception, since their effect was actually decreased by wounding. The proteases operating at pH 4.1 and 6.6 were both clearly less active in the wounded leaves than in controls. The possible mechanism of this inhibitory effect of wounding on senescence is discussed.     

Characterization of leaf senescence and pod development in soybean explants

Excised soybean (Glycine max [L.] Merrill) cv Anoka leaf discs tend to remain green even after the corresponding intact leaves have turned yello on fruiting plants. We have found that explants which include a leaf along with a stem segment (below the node) and one or more pods (maintained on distilled H₂O) show similar but accelerated leaf yellowing and abscission compared with intact plants. In podded explants excised at pre-podfill, the leaves begin to yellow after 16 days, whereas those excised at late podfill begin to yellow after only 6 days. Although stomatal resistances remain low during the first light period after excision, they subsequently increase to levels above those in leaves of intact plants. Explants taken at mid to late podfill with one or more pods per node behave like intact plants in that pod load does not affect the time lag to leaf yellowing. Explant leaf yellowing and abscission are delayed by removal of the pods or seeds or by incubation in complete mineral nutrient solution or in 4.6 micromolar zeatin. Like chorophyll breakdown, protein loss is accelerated in the explants, but minerals or especially zeatin can retard the loss. Pods on explants show rates and patterns of color change (green to yellow to brown) similar to those of pods on intact plants. These changes start earlier in explants on water than in intact plants, but they can be delayed by adding zeatin. Seed dry weight increased in explants, almost as much as in intact plants. Explants appear to be good analogs of the corresponding parts of the intact plant, and they should prove useful for analyzing pod development and mechanisms of foliar senescence. Moreover, our data suggest that the flux of minerals and cytokinin from the roots could influence foliar senescence in soybeans, but increased stomatal resistance does not seem to cause foliar senescence.     

Chilling stress--the key predisposing factor for causing Alternaria alternata infection and leading to cotton (Gossypium hirsutum L.) leaf senescence

Leaf senescence plays a vital role in nutrient recycling and overall capacity to assimilate carbon dioxide. Cotton premature leaf senescence, often accompanied with unexpected short-term low temperature, has been occurring with an increasing frequency in many cotton-growing areas and causes serious reduction in yield and quality of cotton. The key factors for causing and promoting cotton premature leaf senescence are still unclear. In this case, the relationship between the pre-chilling stress and Alternaria alternata infection for causing cotton leaf senescence was investigated under precisely controlled laboratory conditions with four to five leaves stage cotton plants. The results showed short-term chilling stress could cause a certain degree of physiological impairment to cotton leaves, which could be recovered to normal levels in 2-4 days when the chilling stresses were removed. When these chilling stress injured leaves were further inoculated with A. alternata, the pronounced appearance and development of leaf spot disease, and eventually the pronounced symptoms of leaf senescence, occurred on these cotton leaves. The onset of cotton leaf senescence at this condition was also reflected in various physiological indexes such as irreversible increase in malondialdehyde (MDA) content and electrolyte leakage, irreversible decrease in soluble protein content and chlorophyll content, and irreversible damage in leaves' photosynthesis ability. The presented results demonstrated that chilling stress acted as the key predisposing factor for causing A. alternata infection and leading to cotton leaf senescence. It could be expected that the understanding of the key factors causing and promoting cotton leaf senescence would be helpful for taking appropriate management steps to prevent cotton premature leaf senescence.     

Allocation of proteolytic activity in the seedling of Cynara cardunculus L. in the initial growth stages

Cynara cardunculus L. seeds were germinated in vitro under environmentally controlled conditions. Seeds showed a 60% germination rate, and three growth stages were established based on the seedling mean relative growth rate (RGR). Root, stem and cotyledons were compared in these stages with respect to the emergence of total proteases and cardosin activity and its allocation in the seedling. In growth stage I (1st-5th post-germinative days), seedlings grew very slowly. Total proteases and cardosins were already active at the onset of seedlings in the stem. Total soluble protein remained constant in cardoon seedlings during stage I, and the content of all free amino acids (aa) but proline (Pro) was equally allocated on the 1st post-germinative day. In growth stage II (5th-10th post-germinative days), seedlings grew intensively and exhibited fully developed cotyledons. A pronounced increase in the content of all free aa up to the middle of growth stage II in both stems and roots was observed. In addition, the allocation of the total proteolytic activity and cardosins followed a gradient from the root to the seedling shoot. However, the whole seedling soluble protein remained constant up to the 7th day in and tended to peak on the 10th post-germinative day, being allocated mainly to the seedling stem. In growth stage III (10th-15th post-germinative days), cardoon seedlings exhibited the lowest mean RGR and the highest R/S growth ratio. An intensive degradation of total soluble protein present in the whole seedling except for cotyledons (ca. 5-fold) was observed. Nevertheless, in growth stage III, both the gradients exhibited by total proteases and cardosins activities between the root and the seedling shoot were enhanced, as were contents of all aa except Pro, exhibiting the highest levels in cotyledons on the 15th post-germinative day.     

Methyl jasmonate down-regulates endogenous cytokinin levels in cotyledons of Cucurbita pepo (zucchini) seedlings

Endogenous cytokinins (CK) were measured in Cucurbita pepo L. (zucchini) cotyledons after spraying 7-day-old seedlings with methyl jasmonate (MeJA) to examine whether the senescence-promoting action of MeJA is associated with changes in metabolic regulation of CK levels. MeJA promoted senescence as estimated by the loss of chlorophyll and injury to chloroplast ultrastructure. This was already detected one day after treatment. The contents of physiologically active CKs determined by high-performance liquid chromatography-mass spectrometry (CK bases: trans -zeatin, dihydrozeatin and isopentenyladenine and their ribosides), especially trans -zeatin and its riboside declined considerably. CK nucleotides and physiologically inactive CK 7- and 9-glucosides were also markedly decreased whereas the content of storage CK O -glucosides as well as cis -isomers of zeatin declined only slightly. Chlorophyll content and the normal chloroplast structure recovered 5 days after the MeJa treatment. The process of photosynthetic apparatus recovery correlated with an increased concentration of physiologically active CKs, especially Z. In contrast, the content of CK nucleotides and storage CK O -glucosides decreased further. We suggest that the ability of MeJA to promote some aspects of senescence in intact zucchini cotyledons is at least partially due to down-regulation of endogenous CK levels and their interconversion between active and inactive forms.     

Effects of ABA on Senescence and Cellulase Activity of Detached Rice Leaves

The relationship of cellulase to detached leaf senescence of rice seedlings was investigated by examining the effect of ABA and 6-BA on changes in the level of cellulase of leaf segments during senescence. It was shown that the rise in cellulase activity increased with declining chlorophyll content, which was used as the senescence indicator during the senescence of detached rice leaves caused by ABA. The action of ABA took place only after a 48h lag period. ABA enhanced the cellulase secretion and increased the permeability of plasma membrane. A high level of cellulase activity in cell wall closely related to membrane permeability changes. The action of cellulase in the cell wall may cause depolymerization of β-1, 4-glucan in situ, thus speeding senescence. The 6-BA reverses completely or partly the increase in cellulase activity and tile permeability caused by ABA during the first two day, }) ut it antagonized hardly any of the ABA effect from the third day on, suggesting the onset of an irreversible stage in the senescence of detached rice leaves.     

萌发绿豆子叶衰老期间蛋白质代谢的变化

萌发绿豆子叶自然衰老过程中可溶性蛋白质含量一直下降;从衰老开始到衰老前期,总游离氨基酸含量明显上升;但游离氨基酸各组分在子叶衰老期间的变化趋势并不相同。~3H-亮氨酸掺入蛋白质试验和多聚核糖体的相对量及其与总核糖体的比值(P/T)测定都证明在子叶衰老前期有蛋白质的新合成。子叶衰老期间。氨肽酶活性明显降低;而以酪蛋白为底物的蛋白水解酶活性却急剧上升,承担着催化蛋白质降解的主要功能。     

Correlation of xylem sap cytokinin levels with monocarpic senescence in soybean

Cytokinins (CKs) coming from the roots via the xylem are known to delay leaf senescence, and their decline may be important in the senescence of soybean (Glycine max) plants during pod development (monocarpic senescence). Therefore, using radioimmunoassay of highly purified CKs, we quantified the zeatin (Z), zeatin riboside (ZR), the dihydro derivatives (DZ, DZR), the O-glucosides, and DZ nucleotide in xylem sap collected from root stocks under pressure at various stages of pod development. Z, ZR, DZ, and DZR dropped sharply during early pod development to levels below those expected to retard senescence. Pod removal at full extension, which delayed leaf senescence, caused an increase in xylem sap CKs (particularly ZR and DZR), while depodding at late podfill, which did not delay senescence, likewise did not increase the CK levels greatly. The levels of the O-glucosides and the DZ nucleotide were relatively low, and they showed less change with senescence or depodding. The differences in the responses of individual CKs to senescence and depodding suggest differences in their metabolism. Judging from their activity, concentrations and response to depodding, DZR and ZR may be the most important senescence retardants in soybean xylem sap. These data also suggest that the pods can depress CK production by the roots at an early stage and this decrease in CK production is required for monocarpic senescence in soybean.     

Effect of water stress on some oxidative enzymes and senescence in Vigna seedlings

Seedlings of Vigna catjang Endl. were subjected to water stress for 6, S and 10 days by withholding water to investigate the activities of some oxidative enzymes and the pattern of senescence in leaves of 17-day-old seedlings undergoing water stress. Increasing duration of stress produced a proportional increase in the activities of IAA-oxidase, AA-oxidase, peroxidase and glycolate oxidase but decreased catalase activity and the contents of both chlorophyll and protein, hastening senescence. Leaf water potential and relative water content were also lowered with incresing duration of stress. Permeability was increased in leaf tissue undergoing water stress for 8 days. Seed treatment with CaCl₂ (10⁻² and 10⁻¹⁴ M ) for 6 h improved the water status of leaves, decreased tissue permeability, activities of oxidative enzymes, decline of chlorophyll and protein contents and delayed senescence compared to untreated water stressed plants.     

Thigmomorphogenesis: XXIII. Promotion of foliar senescence by mechanical perturbation of Avena sativa and four other species

Mechanical perturbation (MP, gentle tubbing) promoted the senescence of detached oat ( Avena sativa L. cv. Victory) leaf segments in the dark. The promotion of senescence increased with increase in the number of rubbings and could be seen after 24 h of dark incubation; the maximum effect was reached on day 3. The effect (% of control) of MP on the loss of protein was greater than the effect on chlorophyll (Chl) loss on day 1. However, on day 3 the effect of MP on the loss of Chl became greater than the effect on the loss of protein. Ethephon and 1-aminocyclopropane-1-carboxylic acid (ACC) marginally promoted the loss of Chl by both control and rubbed oat leaf segments, and the effect was additive with MP. Chloramphenicol (CAP), spermine, aminoethoxyvinylglycine (AVG) and Ca²⁺ marginally delayed the loss of Chl and protein in both control and rubbed segments. Kinetin greatly retarded the senescence of all segments. Even in the presence of these substances, the amounts of Chl and protein in the rubbed segments were always less than in their respective controls, thus retaining the effect of the MP. However, abscisic acid (ABA) and cycloheximide (CHI) caused the rubbed oat leaf segments to retain more Chl and protein than their respective control segments. The effect of CHI was actually enhanced by MP. Rubbing promoted the senescence of attached leaves of oats ( Avena sativa L. cv. Victory), maize ( Zea mays L. cv. Early Belle) and pumpkin ( Cucurbita pepo L. cv. Jack-o-lantern) cotyledons in the dark. Rubbing promoted the senescence of oat leaf segments even in light, although to a lesser extent compared to the effect in the dark. The senescence of leaves of pumpkin and cocklebur ( Xanthium strumarium Wallr. var. Pennsylvanicum ) in situ was also enhanced by MP.