Increased cytokinin levels in transgenic PSAG12–IPT tobacco plants have large direct and indirect effects on leaf senescence, photosynthesis and N partitioning

We studied the impact of delayed leaf senescence on the functioning of plants growing under conditions of nitrogen remobilization. Interactions between cytokinin metabolism, Rubisco and protein levels, photosynthesis and plant nitrogen partitioning were studied in transgenic tobacco (Nicotiana tabacum L.) plants showing delayed leaf senescence through a novel type of enhanced cytokinin syn‐thesis, i.e. targeted to senescing leaves and negatively auto‐regulated (P_SAG12–IPT), thus preventing developmental abnormalities. Plants were grown with growth‐limiting nitrogen supply. Compared to the wild‐type, endogenous levels of free zeatin (Z)‐ and Z riboside (ZR)‐type cytokinins were increased up to 15‐fold (total ZR up to 100‐fold) in senescing leaves, and twofold in younger leaves of P_SAG12–IPT. In these plants, the senescence‐associated declines in N, protein and Rubisco levels and photosynthesis rates were delayed. Senescing leaves accumulated more (¹⁵N‐labelled) N than younger leaves, associated with reduced shoot N accumulation (–60%) and a partially inverted canopy N profile in P_SAG12–IPT plants. While root N accumulation was not affected, N translocation to non‐senescing leaves was progressively reduced. We discuss potential consequences of these modified sink–source relations, associated with delayed leaf senescence, for plant productivity and the efficiency of utilization of light and minerals.     

Effects of cytokinin and senescence-inducing factors on expression of P ARR5 -GUS gene construct during leaf senescence in transgenic Arabidopsis thaliana plants

ARR5-gene expression was studied in the course of natural leaf senescence and detached leaf senescence in the dark using Arabidopsis thaliana plants transformed with the P _ARR5 -GUS gene construct. GUS-activity was measured as a marker of ARR5-gene expression. Chlorophyll and total protein amounts were also estimated to evaluate leaf senescence. Natural leaf senescence was accompanied by the progressive decline in the GUS-activity in leaves of the 2nd and 3rd nodes studied, and this shift of GUS-activity was more pronounced than the loss of chlorophyll content. The ability of the ARR5-gene promoter to respond to cytokinin was not eliminated during natural leaf senescence, as was demonstrated by a cytokinin-induced increase in GUS activity in leaves after their detachment and incubation on benzyladenine (BA, 5 × 10⁻⁶ M) in the dark. Leaf senescence in the dark was associated with the further decrease in the GUS-activity. The ARR5-gene promoter response to cytokinin was enhanced with the increase of the age of plants, taken as a source of leaves for cytokinin treatments. Hence, although the expression of the ARR5 gene reduces during natural and dark/detached leaf senescence, the ARR5-gene sensitivity to cytokinin was maintained in both cases and even increased with the leaf age. This data suggest that the ARR5 gene, which belongs to the type-A negative regulators of plant response to cytokinin, could be a feedback regulator able to prevent retardation by cytokinin of leaf senescence when it is important for plant life. Growth regulators either reduced ARR5 gene response to cytokinin during senescence of mature detached leaves in the dark (SA, meJA, ABA, SP) or increased it (IAA), thus modifying the resulting rate of its expression.     

Leaf senescence is delayed in tobacco plants expressing the maize homeobox gene knotted1 under the control of a senescence-activated promoter.

Leaf senescence is an active process involving remobilization of nutrients from senescing leaves to other parts of the plant. Whereas senescence is accompanied by a decline in leaf cytokinin content, supplemental cytokinin delays senescence. Plants that overexpress isopentenyl transferase (ipt), a cytokinin-producing gene, or knotted1 (kn1), a homeobox gene, have many phenotypes in common. Many of these phenotypes are characteristic of altered cytokinin physiology. The effect of kn1 on leaf senescence was tested by driving its expression using the promoter of the senescence-associated gene SAG12. SAG:kn1 tobacco plants showed a marked delay in leaf senescence but otherwise developed normally. The delay in senescence was revealed by an increase in chlorophyll content in SAG:kn1 leaves relative to leaves of the control plants and by a decrease in the number of dead leaves. Senescence was also delayed in detached leaves of SAG:kn1 plants. Delayed senescence was accompanied by increased leaf cytokinin content in older leaves expressing kn1. These experiments extend the current understanding of kn1 function and suggest that in addition to mediating meristem maintenance, kn1 is capable of regulating the onset of senescence in leaves.     

Development of flooding-tolerant Arabidopsis thaliana by autoregulated cytokinin production

Flooding is one of the most serious environmental stresses that affect plant growth and productivity. Flooding causes premature senescence which results in leaf chlorosis, necrosis, defoliation, cessation of growth and reduced yield. This study was conducted to determine the effects of autoregulated cytokinin production on the flooding tolerance of Arabidopsis thaliana plants. A chimeric gene containing the senescence-specific SAG12 promoter and the ipt gene coding for isopentenyl transferase, a rate-limiting enzyme in the cytokinin biosynthesis pathway, was constructed. The chimeric gene was introduced into Arabidopsis plants by Agrobacterium-mediated vacuum infiltration. Four transgenic lines were chosen for flooding tolerance determinations. DNA hybridization analysis and PCR confirmed that all four of the transgenic lines carried the ipt gene. The segregation of kanamycin resistance in the T₂ generation indicated 1 to 3 integration events. GUS expression and RT-PCR of the ipt gene confirmed the senescence-specificity of the SAG12 promoter. Morphologically, the transgenic lines appeared healthy and normal. Transgenic plants began to flower at the same time as wild-type plants, but the period from flowering to senescence was lengthened by 7 to 12 days. Tolerance of the transgenic plants to waterlogging and complete submergence was assayed in three independent experiments. All four transgenic lines were consistently more tolerant to flooding than wild-type plants. The results indicated that endogenously produced cytokinin can regulate senescence caused by flooding stress, thereby, increasing plant tolerance to flooding. This study provides a novel mechanism to improve flooding tolerance in plants.     

Light Restriction Delays Leaf Senescence in Winter Oilseed Rape (Brassica napus L.)

Oilseed rape (Brassica napus L.) is a crop with a complex aerial architecture that can cause self-shading leading to a vertical light gradient over the foliage. Mutual shading between neighboring plants at a high sowing density also results in an alteration of photosynthetically active radiation (PAR) absorption by lower leaves. The aim of this study was to analyze the impact that light restriction on lower leaves has on shoot architecture, biomass production and allocation, nitrogen (N) fluxes, and progression of sequential senescence. Field-grown plants were collected at the end of the vegetative rest period and grown in hydroponic conditions until pod maturity. A shading treatment corresponding to a 43.4 % reduction of PAR was applied at the early flowering stage. N uptake and fluxes of N allocation and remobilization were determined by supplying K¹⁵NO₃ in the nutrient solution. Photosynthesis and expression of SAG12 and Cab genes (indicators of leaf senescence progression) were also analyzed on different leaf ranks. The results showed that shading enhanced leaf development on the main stem and ramifications to optimize light capture. The expression pattern of the SAG12/Cab molecular indicator suggested a delay in leaf senescence that allowed leaf life span to be extended resulting in a more efficient leaf compound remobilization, with lower N residual contents in fallen leaves under shading. N uptake increased and N remobilization fluxes were enhanced from source organs (leaves and stem) toward sink organs (flowers). Profuse branching and late senescing varieties would be of interest for further selection programs under high sowing densities.     

Transgenic rice with low sucrose-phosphate synthase activities retain more soluble protein and chlorophyll during flag leaf senescence

We investigated whether changes in sucrose-phosphate synthase (EC 2.4.1.14, SPS) activity could alter N remobilization during leaf senescence. Transgenic rice (Oryza sativa L. cv. Nipponbare) with low SPS activities and wild-type rice plants were grown with basal N (1.0 mM NH₄NO₃) until the late vegetative stage. Subsequently, half of the plants were transferred to a low N (0.1 mM NH₄NO₃) condition to accelerate leaf senescence, and the others were continuously grown with basal N. With low N supply, the amounts of chlorophyll and soluble protein in flag leaf blades decreased after anthesis in both the low SPS plants and wild-type plants, although the decrease was less in the low SPS plants. Panicle weights were significantly lower in the low SPS plant than in the wild-type plant. These results suggest that the remobilization of N from flag leaves was diminished by suppressing the development of reproductive sinks in the low SPS plant.     

Modification of senescence in ryegrass transformed with IPT under the control of a monocot senescence-enhanced promoter

We report here the genetic modification of ryegrass senescence. Embryogenic cell suspensions of Lolium multiflorum were transformed by microprojectile bombardment with plasmid constructs containing 1.98 kb of the 5 flanking sequence of SEE1 (a maize cysteine protease gene showing enhanced expression during senescence) fused either to the Agrobacterium tumefaciens cytokinin biosynthesis gene IPT (designated P_SEE1::IPT) or to the -glucuronidase reporter gene UIDA (P_SEE1::UIDA). Plants were regenerated under selection for the HPH hygromycin resistance gene in the vector. P_SEE1::UIDA transformants confirmed that the SEE1 flanking sequence functioned as a senescence-enhanced promoter in ryegrass. The IPT transgene was detected in 28 regenerants (P_SEE1::IPT) from five independent transformation events. P_SEE1::IPT leaves displayed a stay-green phenotype. Some P_SEE1::IPT lines developed spontaneous lesions.Abbreviations Chl: Chlorophyll - GUS: -Glucuronidase - RChl: Relative Chl content - WT: Wild-typeCommunicated by M.R. Davey     

Growth response of barley and tomato to nitrogen stress and its control by abscisic acid,water relations and photosynthesis

Barley (Hordeum vulgare L.) and tomato Lycopersicon esculentum Mill.) were grown hydroponically and examined 2, 5, and 10 d after being deprived of nitrogen (N) supply. Leaf elongation rate declined in both species in response to N stress before there was any reduction in rate of dryweight accumulation. Changes in water transport to the shoot could not explain reduced leaf elongation in tomato because leaf water content and water potential were unaffected by N stress at the time leaf elongation began to decline. Tomato maintained its shoot water status in N-stressed plants, despite reduced water absorption per gram root, because the decline in root hydraulic conductance with N stress was matched by a decline in stomatal conductance. In barley the decline in leaf elongation coincided with a small (8%) decline in water content per unit area of young leaves; this decline occurred because root hydraulic conductance was reduced more strongly by N stress than was stomatal conductance. Nitrogen stress caused a rapid decline in tissue NO ₃ ^- pools and in NO ₃ ^- flux to the xylem, particularly in tomato which had smaller tissue NO ₃ ^- reserves. Even in barley, tissue NO ₃ ^- reserves were too small and were mobilized too slowly (60% in 2 d) to support maximal growth for more than a few hours. Organic N mobilized from old leaves provided an additional N source to support continued growth of N-stressed plants. Abscisic acid (ABA) levels increased in leaves of both species within 2 d in response to N stress. Addition of ABA to roots caused an increase in volume of xylem exudate but had no effect upon NO ₃ ^- flux to the xylem. After leaf-elongation rate had been reduced by N stress, photosynthesis declined in both barley and tomato. This decline was associated with increased leaf ABA content, reduced stomatal conductance and a decrease in organic N content. We suggest that N stress reduces growth by several mechanisms operating on different time scales: (1) increased leaf ABA content causing reduced cell-wall extensibility and leaf elongation and (2) a more gradual decline in photosynthesis caused by ABA-induced stomatal closure and by a decrease in leaf organic N.Abbreviation and symbols ABA abscisic acid - c_i leaf internal CO₂ concentration - L_p root hydraulic conductance     

The <Emphasis Type="Italic">ABI2</Emphasis>-dependent abscisic acid signalling controls HrpN-induced drought tolerance in <Emphasis Type="Italic">Arabidopsis</Emphasis>

HrpN, a protein produced by the plant pathogenic bacterium Erwinia amylovora, has been shown to stimulate plant growth and resistance to pathogens and insects. Here we report that HrpN activates abscisic acid (ABA) signalling to induce drought tolerance (DT) in Arabidopsis thaliana L. plants grown with water stress. Spraying wild-type plants with HrpN-promoted stomatal closure decreased leaf transpiration rate, increased moisture and proline levels in leaves, and alleviated extents of damage to cell membranes and plant drought symptoms caused by water deficiency. In plants treated with HrpN, ABA levels increased; expression of several ABA-signalling regulatory genes and the important effector gene rd29B was induced or enhanced. Induced expression of rd29B, promotion of stomatal closure, and reduction in drought severity were observed in the abi1-1 mutant, which has a defect in the phosphatase ABI1, after HrpN was applied. In contrast, HrpN failed to induce these responses in the abi2-1 mutant, which is impaired in the phosphatase ABI2. Inhibiting wild-type plants to synthesize ABA eliminated the role of HrpN in promoting stomatal closure and reducing drought severity. Moreover, resistance to Pseudomonas syringae developed in abi2-1 as in wild-type plants following treatment with HrpN. Thus, an ABI2-dependent ABA signalling pathway is responsible for the induction of DT but does not affect pathogen defence under the circumstances of this study.Hong-Ping Dong and Haiqin Yu contributed equally to this study and are regarded as joint first authors.     

Root Growth of Arabidopsis thaliana Is Regulated by Ethylene and Abscisic Acid Signaling Interaction in Response to HrpNEa, a Bacterial Protein of Harpin Group

HrpN_Ea is a harpin protein from Erwinia amylovora, a bacterial pathogen that causes fire blight in rosaceous plants. Treating plants with HrpN_Ea stimulates ethylene and abscisic acid (ABA) to induce plant growth and drought tolerance, respectively. Herein, we report that both growth hormones cooperate to mediate the role of HrpN_Ea in promoting root growth of Arabidopsis thaliana seedlings. Root growth is promoted coordinately with elevation in levels of ABA and ethylene subsequent to soaking of germinating seeds of wild-type (WT) Arabidopsis in a solution of HrpN_Ea. However, these responses are arrested by inhibiting WT roots from synthesizing ethylene as well as sensing of ABA and ethylene. The effects of HrpN_Ea on roots are also nullified in ethylene-insensitive etr1-1 and ein5-1 mutants and in the ABA-insensitive mutant abi2-1 of Arabidopsis. These results provide evidence for presence of a relationship between root growth enhancement and signaling by ABA and ethylene in response to HrpN_Ea. Nevertheless, when HrpN_Ea is applied to leaves, ethylene signaling is active in the absence of ABA signaling to promote plant growth. This suggests the presence of a different signaling mechanism in leaves from that in roots. X. Ren and F. Liu contributed equally to this study and are regarded as joint first authors     

The impact of <Emphasis Type="Italic">trans</Emphasis>-zeatin <Emphasis Type="Italic">O</Emphasis>-glucosyltransferase gene over-expression in tobacco on pigment content and gas exchange

The responses of tobacco plants over-expressing trans-zeatin O-glucosyltransferase gene under constitutive or senescence-inducible promoter (35S:ZOG1 and SAG12:ZOG1) and of wild type (WT) plants to water stress and subsequent rehydration were compared. In plants sufficiently supplied with water, both transgenics have higher net photosynthetic rate (P_N) in upper and middle leaves and higher stomatal conductance (g_s) in middle leaves than WT. Water use efficiency (WUE = P_N/E) was higher in both transgenics than in WT. During prolonged water stress, both P_N and E declined to a similar extent in both transgenics and WT plants. However, 7 d after rehydration P_N in SAG:ZOG (upper and middle leaves) and 35S:ZOG (upper leaves) was higher than that in WT plants. Increased content of endogenous CKs in 35S:ZOG plants did not prevent their response to ABA application and the results obtained did not support concept of CK antagonism of ABA-induced stomatal closure. The chlorophyll (Chl) a+b content was mostly higher in both transgenics than in WT. During water stress and subsequent rehydration it remained unchanged in upper leaves, decreased slightly in middle leaves only of WT, while rapidly in lower leaves. Total degradation of Chl, carotenoids and xanthophyll cycle pigments (XCP) was found under severe water stress in lower leaves. Carotenoid and XCP contents in middle and upper leaves mostly increased during development of water stress and decreased after rehydration. While β-carotene content was mostly higher in WT, neoxanthin content was higher in transgenics especially in 35S:ZOG under severe stress and after rehydration. The higher content of XCP and degree of their deepoxidation were usually found in upper and middle leaves than in lower leaves with exception of SAG:ZOG plants during mild water stress.     

Regulated Expression of a Cytokinin Biosynthesis Gene IPT Delays Leaf Senescence and Improves Yield under Rainfed and Irrigated Conditions in Canola (Brassica napus L.)

Delay of leaf senescence through genetic modification can potentially improve crop yield, through maintenance of photosynthetically active leaves for a longer period. Plant growth hormones such as cytokinin regulate and delay leaf senescence. Here, the structural gene (IPT) encoding the cytokinin biosynthetic enzyme isopentenyltransferase was fused to a functionally active fragment of the AtMYB32 promoter and was transformed into canola plants. Expression of the AtMYB32xs::IPT gene cassette delayed the leaf senescence in transgenic plants grown under controlled environment conditions and field experiments conducted for a single season at two geographic locations. The transgenic canola plants retained higher chlorophyll levels for an extended period and produced significantly higher seed yield with similar growth and phenology compared to wild type and null control plants under rainfed and irrigated treatments. The yield increase in transgenic plants was in the range of 16% to 23% and 7% to 16% under rainfed and irrigated conditions, respectively, compared to control plants. Most of the seed quality parameters in transgenic plants were similar, and with elevated oleic acid content in all transgenic lines and higher oil content and lower glucosinolate content in one specific transgenic line as compared to control plants. The results suggest that by delaying leaf senescence using the AtMYB32xs::IPT technology, productivity in crop plants can be improved under water stress and well-watered conditions.     

The efficiency of water use in water stressed plants is increased due to ABA induced stomatal closure

Gas exchange and abscisic acid content of Digitalis lanata EHRH. have been examined at different levels of plant water stress. Net photosynthesis, transpiration and conductance of attached leaves declined rapidly at first, then more slowly following the withholding of irrigation. The intercellular partial pressure of CO₂ decreased slightly. The concentration of 2-cis(S)ABA increased about eight-fold in the leaves of non-irrigated plants as compared with well-watered controls. A close linear correlation was found between the ABA content of the leaves and their conductance on a leaf area basis. In contrast, the plot of net assimilation versus ABA concentration was curvilinear, leading to an increased efficiency of water use during stress. After rewatering, photosynthesis reached control values earlier than transpiration, leaf conductance and ABA content. From these data it is concluded that transpiration through the stomata is directly controlled by the ABA content, whereas net photosynthesis is influenced additionally by other factors.Possible reasons for the responses of photosynthesis and water use efficiency to different stress and ABA levels are discussed.Abbreviations A net CO₂ assimilation - ABA abscisic acid - C_i intercellular CO₂ concentration - g stomatal conductance - T transpiration - WUE water use efficiency     

Overexpressing <Emphasis Type="Italic">SgNCED1</Emphasis> in Tobacco Increases ABA Level,Antioxidant Enzyme Activities,and Stress Tolerance

Abscisic acid (ABA) regulates plant adaptive responses to various environmental stresses. 9-cis-epoxycarotenoid dioxygenase (NCED) is the key enzyme of ABA biosynthesis in higher plants. A NCED gene, SgNCED1, was overexpressed in transgenic tobacco plants which resulted in 51–77% more accumulation of ABA in leaves. Transgenic tobacco plants decreased stomatal conductance, transpiration rate, and photosynthetic rate but induced activities of superoxide dismutase (SOD), catalase (CAT), and ascorbate-peroxidase (APX). Hydrogen peroxide (H₂O₂) and nitric oxide (NO) in leaves were also induced in the transgenic plants. Compared to the wild-type control, the transgenic plants improved growth under 0.1 M mannitol-induced drought stress and 0.1 M NaCl-induced salinity stress. It is suggested that the ABA-induced H₂O₂ and NO generation upregulates the stomatal closure and antioxidant enzymes, and therefore increases drought and salinity tolerance in the transgenic plants.     

Cross-talks between Ca<Superscript>2+</Superscript>/CaM and H<Subscript>2</Subscript>O<Subscript>2</Subscript> in abscisic acid-induced antioxidant defense in leaves of maize plants exposed to water stress

Here we examined whether Ca²⁺/Calmodulin (CaM) is involved in abscisic acid (ABA)-induced antioxidant defense and the possible relationship between CaM and H₂O₂ in ABA signaling in leaves of maize (Zea mays L.) plants exposed to water stress. An ABA-deficient mutant vp5 and its wild type were used for the experimentation. We found that water stress enhanced significantly the contents of CaM and H₂O₂, and the activities of chloroplastic and cytosolic superoxide dismutase (SOD), ascorbate peroxidase (APX) and glutathione reductase (GR), and the gene expressions of the CaM1, cAPX, GR1 and SOD4 in leaves of wild-type maize. However, the increases mentioned above were almost arrested in vp5 plants and in the wild-type plants pretreated with ABA biosynthesis inhibitor tungstate (T), suggesting that ABA is required for water stress-induced H₂O₂ production, the enhancement of CaM content and antioxidant defense. Besides, we showed that the up-regulation of water stress-induced antioxidant defense was almost completely blocked by pretreatment with Ca²⁺ inhibitors, CaM antagonists and reactive oxygen (ROS) manipulators. Moreover, the analysis of time course of CaM and H₂O₂ production under water stress showed that the increase in CaM content preceded that of H₂O₂. These results suggested that Ca²⁺/CaM and H₂O₂ were involved in the ABA-induced antioxidant defense under water stress, and the increases of Ca²⁺/CaM contents triggered H₂O₂ production, which inversely affected the contents of CaM. Thus, a cross-talk between Ca²⁺/CaM and H₂O₂ may play a pivotal role in the ABA signaling.     

Low sink-induced stomatal closure alters photosynthetic rates of source leaves in beans as dependent on H2O2 and ABA accumulation in guard cells

Low sink demand provided by pod removal and stem girdling of beans (Vicia faba, cv. Daqingshan) (-Sink) induced a significantly lower net photosynthetic rate (P _n), stomatal conductance (g _s), internal CO₂ concentration (C _i), and transpiration rate (E) compared with pod and root sink retention (CK). This depression in P _n was due to stomatal limitation. Low sink demand of -Sink plants resulted in a higher leaf sucrose content, but a lower sucrose content in guard cells. Moreover, the significant accumulation of H₂O₂ and ABA were observed in both leaves and guard cells of -Sink plants. The most intensive electron dense deposit of cerium perhydroxides, produced by H₂O₂ reaction with cerium chloride, was present in the cell walls, especially the dorsal walls of guard cells. Immunogold electron-microscopy localization of ABA showed that ABA was distributed in ventral walls of guard cells and the intercellular space of mesophyll cells of -Sink leaves in contrast to CK plants. Application of exogenous sucrose to isolated bean leaves increased H₂O₂ and ABA contents. H₂O₂ and ABA in leaves was likely generated by two independently regulated pathways, each affected by the high sucrose concentration induced by low sink demand. Increased sucrose in leaves in response to low sink demand may have caused the increase of H₂O₂ and ABA, and their accumulation in mesophyll cells and guard cells was likely the primary cause for stomatal closure under low sink demand.     

Overexpression of Farnesyl Diphosphate Synthase in Arabidopsis Mitochondria Triggers Light-dependent Lesion Formation and Alters Cytokinin Homeostasis

To investigate the role of mitochondrial farnesyl diphosphate synthase (FPS) in plant isoprenoid biosynthesis we characterized transgenic Arabidopsis thaliana plants overexpressing FPS1L isoform. This overexpressed protein was properly targeted to mitochondria yielding a mature and active form of the enzyme of 40 kDa. Leaves from transgenic plants grown under continuous light exhibited symptoms of chlorosis and cell death correlating to H₂O₂ accumulation, and leaves detached from the same plants displayed accelerated senescence. Overexpression of FPS in mitochondria also led to altered leaf cytokinin profile, with a reduction in the contents of physiologically active trans-zeatin- and isopentenyladenine-type cytokinins and their corresponding riboside monophosphates as well as enhanced levels of cis-zeatin 7-glucoside and storage cytokinin O-glucosides. Overexpression of 3-hydroxy-3-methylglutaryl coenzyme A reductase did not prevent chlorosis in plants overexpressing FPS1L, but did rescue accelerated senescence of detached leaves and restored wild-type levels of cytokinins. We propose that the overexpression of FPS1L leads to an enhanced uptake and metabolism of mevalonic acid-derived isopentenyl diphosphate and/or dimethylallyl diphosphate by mitochondria, thereby altering cytokinin homeostasis and causing a mitochondrial dysfunction that renders plants more sensitive to the oxidative stress induced by continuous light.     

The relationship between stomatal resistance and abscisic-acid levels in leaves of water-stressed bean plants

Leaf water potentials of Phaseolus vulgaris L. plants exposed to a -3.0 bar root medium were reduced to between -7 and -9 bars within 25 min and remained constant for the next several hours. This treatment led to considerable variation between leaves in both abscisic-acid (ABA) content and R_s, although the two were well correlated after a 5-h treatment. There was an apparent 7-fold increase in leaf ABA levels necessary to initiate stomatal closure when plants were exposed to a -3.0 bar treatment, but when plants were exposed to a -5.0 bar stress R_s values increased prior to any detectable rise in ABA levels. To explain these seemingly contradictory results, we suggest that the rate of ABA synthesis in the leaf, rather than the total ABA content, determines the status of the stomatal aperture.Abbreviations ABA abscisic acid - PEG polyethylene glycol - R_s stomatal diffusion resistance of lower leaf surface - leaf water potential     

The Effects of Fusicoccin,Abscisic Acid,and Benzyladenine on the Transport and Partitioning of Substances as Related to Cytokinin Sink Activity

We tested the possible cytokinin effect on the functioning of the active transport system involved in the assimilate loading into the phloem as a cause for the cytokinin sink and retention effect. This effect is manifested in the deceleration of substance export from and the stimulation of substance import to the sites of local phytohormone application to the mature detached leaf from untreated leaf areas. To affect the membrane mechanisms of the substance transport, we used leaf treatment with the phytotoxin fusicoccin, an enhancer of plasmalemmal H⁺-ATPase and a potential stimulator of assimilates export, and with the phytohormone ABA affecting transport, metabolism, and plant growth. However, fusicoccin did not enhance ¹⁴C-sucrose export from the leaf blade and did not interfere with the cytokinin-induced export deceleration. ABA reduced substantially ¹⁴C export from the leaf but eliminated the cytokinin effect on this process. Similar results were obtained for broad bean (Vicia faba L.) leaves with apoplastic phloem loading, involving H⁺-ATPase activity, and pumpkin (Cucurbita pepo L.) leaves with symplastic phloem loading, that is, occurring without sucrose transmembrane translocation and without H⁺-ATPase involvement. The conclusion is that the cytokinin-induced development of sink zones in source leaves is not related to the membrane mechanisms of the substance transport in the mesophyll–phloem system. The data obtained support the idea that the cause for the cytokinin sink and retention effect is the enhancement of elongation growth and total activation of metabolism in the mesophyll cells of the detached leaf.