Cytokinin (CK) levels in cotyledons of Cucurbita pepo L. (zucchini) were investigated through the processes of postgermination, 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.Abbreviations -CK, cytokinin; DHZ, dihydrozeatin; DHZ7G, dihydrozeatin 7-glucoside; DHZ9G, dihydrozeatin 9-glucoside; DHZR, dihydrozeatin 9-riboside; DHZRMP, dihydrozeatin 9-riboside-5 0 -monophosphate; DHZROG, dihydrozeatin 9-riboside O-glucoside; FW, fresh weight; iP, N 6 -(D 2 -isopentenyl)adenine; iP7G, N 6 -(D 2 -isopentenyl)adenine 7-glucoside; iP9G, N 6 -(D 2 -isopentenyl)adenine 9-glucoside; iPR, N 6 -(D 2 -isopentenyl)adenine 9-riboside; iPRMP, N 6 -(D 2 -isopentenyl)adenine 9-riboside-5 0

Darkness mediates different senescence-related responses depending on the targeting of dark treatment (whole plants or individual leaves) and on the organs that perceive the signal (leaves or cotyledons). As no data are available on the potential role of darkness to promote senescence when applied to individual cotyledons, we have investigated how darkness affects the progression of senescence in either a single or both individually darkened cotyledons of young 10-day-old Cucurbita pepo (zucchini) seedlings. Strong acceleration of senescence was observed when both cotyledons were darkened as judged by the damage in their anatomical structure, deterioration of chloroplast ultrastructure in parallel with decreased photosynthetic rate and photochemical quantum efficiency of PSII. In addition, the endogenous levels of cytokinins (CKs) and IAA were strongly reduced. In a single individually darkened cotyledon, the structure and function of the photosynthetic apparatus as well as the contents of endogenous CKs and IAA were much less affected by darkness, thus suggesting inhibitory effect of the illuminated cotyledon on the senescence of the darkened one. Apparently, the effect of darkness to accelerate/delay senescence in a single darkened cotyledon depends on the light status of the other cotyledon from the pair. The close positive correlation between CK content and the activity of CK oxidase/dehydrogenase (CKX; EC 1.4.3.18/1.5.99.12) suggested that CKX was essentially involved in the mechanisms of downregulation of endogenous CK levels. Our results indicated that CKX-regulated CK signaling could be a possible regulatory mechanism controlling senescence in individually darkened cotyledons.

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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.

Natural senescence of Cucurbita pepo (zucchini) cotyledons was accompanied by a gradual degradation of reserve proteins (globulins) and an intensive decrease in the content of both large subunit (LSU) and small subunit (SSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The net photosynthetic rate, the primary photochemical activity of PSII, estimated by the variable fluorescence (F v )/maximal fluorescence (F m ) ratio (F v /F m ) and the actual quantum yield of PSII electron transport in the light-adapted state (UPSII) also progressively decreased during natural senescence. In contrast, the fraction of the absorbed light energy, which is not used for photochemistry (LNU) increased with progression of senescence. The decline in the photosynthetic rate started earlier in ontogenesis compared with the down-regulation of the functional activity of PSII, thus suggesting the existence of protective mechanisms which maintain higher efficiency of the photochemical electron transport reactions of photosynthesis compared with the dark reactions of the Calvin cycle during earlier stages of natural senescence. Decapitation of the epicotyl above the senescing cotyledons resulted in full recovery of the polypeptide profile in the rejuvenated cotyledons. In addition, the photosynthetic rate increased reaching values that exceeded those measured in juvenile cotyledons. The photochemical efficiency of PSII also gradually recovered, although it did not reach the maximum values measured in the presenescent cotyledons. KeywordsCotyledons of Cucurbita pepo L. (zucchini) Á Natural senescence Á Photosynthesis Á Polypeptide pattern Á Rejuvenation Abbreviations PSIIPhotosystem II UPSIIThe actual quantum yield of PSII electron transport in the light-adapted state LSU

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Darkness mediates different senescencerelated responses depending on the targeting of dark treatment (whole plants or individual leaves) and the organs that perceive the signal (leaves or cotyledons). In this study, we presented data on the differential effects of 2-day dark treatment on progression of senescence in cotyledons and primary leaves of 14-day-old plants of Cucurbita pepo L. (zucchini). The lack of changes in chlorophyll a, chlorophyll b and carotenoid contents as well as in the PSI activity measured by the far-red induced alterations in the P 700 oxidation levels and the quantum yield of electron transport from Q A -to PSI end acceptors (u R 0 ) indicated higher resistance of cotyledons to the applied dark stress compared to the primary leaves. In contrast to cotyledons, PSI activity in the primary leaves was significantly inhibited. Concerning the activity of PSII analyzed by the changes in the JIPtest parameters (the maximal efficiency of PSII photochemistry, u P 0 ; the performance index, PI ABS ; the efficiency of Q A -reoxidation, w 0 and the effective dissipation per reaction center, DI 0 /RC), no differences were observed between cotyledons and primary leaves, thus suggesting that PSI activity in the true leaves was more susceptible to the applied dark stress. The transfer of the darkened plants to normal light regime resulted in delayed senescence in cotyledons which was in contrast to results on Arabidopsis, thus implying the existence of specific mechanisms of cotyledon senescence in different monocarpic plants.Keywords Dark-induced senescence and recovery Á Cotyledons and primary leaves Á PSII and PSI activity Á JIP-test Abbreviations PSA Photosynthetic apparatus PSI Photosystem I PSII Photosystem II RC PSII reaction center Chl

The aim of the present work was to study the response of two sunflower genotypes (cultivated sunflower Helianthus annuus cv. 1114 and newly developed genotype H. annuus × Helianthus argophyllus) to Pb medium-term stress and the role of exogenously applied EDTA in alleviating Pb toxicity in hydroponics. Plant growth, morpho-anatomical characteristics of the leaf tissues, electrolyte leakage, total antioxidant activity, free radical scavenging capacity, total flavonoid content, and superoxide dismutase isoenzyme profile were studied by conventional methods. Differential responses of both genotypes to Pb supplied in the nutrient solution were recorded. Pb treatment induced a decrease in the relative growth rate, disturbance of plasma membrane integrity, and changes in the morpho-anatomical characteristics of the leaf tissues and in the antioxidant capacity, which were more pronounced in the cultivated sunflower H. annuus cv. 1114. The new genotype demonstrated higher tolerance to Pb when compared with the cultivar. This was mainly due to increased photosynthetically active area, maintenance of plasma membrane integrity, permanently high total antioxidant activity, and free radical scavenging capacity as well as total flavonoid content. The addition of EDTA into the nutrient solution led to limitation of the negative impact of Pb ions on the above parameters in both genotypes. This could be related to the reduced content of Pb in the roots, stems, and leaves, suggesting that the presence of EDTA limited the uptake of Pb. The comparative analysis of the responses to Pb treatment showed that the deleterious effect of Pb was more pronounced in the cultivated sunflower H. annuus cv. 1114. The new genotype H. annuus × H. argophyllus was more productive and demonstrated higher tolerance to Pb medium-term stress, which could indicate that it may possess certain mechanisms to tolerate high Pb concentrations. This character could be inherited from the wild parent used in the interspecific hybridization. The ability of EDTA to prevent Pb absorption by the plants could underly the mechanism of limiting of the negative impact of Pb ions. Hence, EDTA cannot be used to enhance Pb absorption from nutrient solution by sunflower plants for phytoremediation purposes.