Cytokinins in plant senescence: From spray and pray to clone and play

Three approaches have been used to investigate the inhibitory role of the cytokinin class of phytohormones in plant senescence: external application of cytokinins, measurement of endogenous cytokinin levels before and during senescence, and manipulation of endogenous cytokinin production in transgenic plants. In transgenic plant studies, endogenous cytokinin levels are manipulated by expression of IPT, a gene encoding isopentenyl transferase. Transgenic plants expressing IPT from a variety of promoters exhibit developmental and morphological alterations and often display retarded leaf senescence. A recently developed autoregulatory senescence-inhibition system targets cytokinin production quantitatively, spatially and temporally, and results in transgenic plants that exhibit significantly delayed senescence without abnormalities. These transgenic studies not only confirm the regulatory role of cytokinins in plant senescence, but also provide a way to manipulate senescence for potential agricultural applications.     

Alterations of Endogenous Cytokinins in Transgenic Plants Using a Chimeric Isopentenyl Transferase Gene

Cytokinins, a class of phytohormones, appear to play an important role in the processes of plant development. We genetically engineered the Agrobacterium tumefaciens isopentenyl transferase gene, placing it under control of a heat-inducible promoter (maize hsp70). The chimeric hsp70 isopentenyl transferase gene was transferred to tobacco and Arabidopsis plants. Heat induction of transgenic plants caused the isopentenyl transferase mRNA to accumulate and increased the level of zeatin 52-fold, zeatin riboside 23-fold, and zeatin riboside 5[prime]-monophosphate twofold. At the control temperature zeatin riboside and zeatin riboside 5[prime]-monophosphate in transgenic plants accumulated to levels 3 and 7 times, respectively, over levels in wild-type plants. This uninduced cytokinin increase affected various aspects of development. In tobacco, these effects included release of axillary buds, reduced stem and leaf area, and an underdeveloped root system. In Arabidopsis, reduction of root growth was also found. However, neither tobacco nor Arabidopsis transgenic plants showed any differences relative to wild-type plants in time of flowering. Unexpectedly, heat induction of cytokinins in transgenic plants produced no changes beyond those seen in the uninduced state. The lack of effect from heat-induced increases could be a result of the transient increases in cytokinin levels, direct or indirect induction of negating factor(s), or lack of a corresponding level of competent cellular factors. Overall, the effects of the increased levels of endogenous cytokinins in non-heat-shocked transgenic plants seemed to be confined to aspects of growth rather than differentiation. Since no alterations in the programmed differentiation pattern were found with increased cytokinin levels, this process may be controlled by components other than absolute cytokinin levels.     

Antioxidant enzymatic protection during tobacco leaf ageing is affected by cytokinin depletion

Plant ageing and senescence are associated with increased levels of reactive oxygen species. Level of cytokinins, the apparent inhibitors of plant senescence, is controlled by their irreversible degradation catalysed by cytokinin oxidase/dehydrogenase (CKX). We investigated the CKX activity, cytokinin concentration, and activities of antioxidative enzymes in tobacco (Nicotiana tabacum L. cv. Samsun NN) overexpressing the Arabidopsis gene for AtCKX2, targeted for extracellular secretion pathway. The control and AtCKX2 plants differed substantially in their phenotypes. When the lowest leaves in controls became yellow all leaves in AtCKX2 tobacco still remained green. Activities of antioxidant enzymes decreased with leaf age in both tobacco plants except for ascorbate peroxidase (APX) in the old leaves and glutathione reductase (GR) in young leaves. Enhancement of GR activity at all leaf stages, an increase of superoxide dismutase and a decline of catalase in young leaves, as well as an increase of APX in the oldest leaves were observed in AtCKX2 plant compared to control. Similar changes were detected after determination of isoenzymes on zymograms. It is evident that AtCKX2 plants had postponed onset of senescence despite the significantly lowered level of cytokinins. Enhanced antioxidant protection, especially in the oldest leaves, could subsidise this phenomenon.     

细胞分裂素对植物衰老的延缓作用

细胞分裂素是一类重要的植物激素,它可在一定程度上延缓植物的衰老。主要从3个方面综述了细胞分裂素与植物衰老之间的关系,即:(1)植物衰老过程中内源细胞分裂素含量变化;(2)外源细胞分裂素的影响;(3)转入与细胞分裂素的合成、降解相关的基因对植物衰老产生的影响。此外,还从细胞分裂素与糖、与脂质氧化反应以及与其它植物激素的关系方面探讨了细胞分裂素在延缓植物衰老中的作用机理。     

Extracellular invertase is an essential component of cytokinin-mediated delay of senescence

Leaf senescence is the final stage of leaf development in which the nutrients invested in the leaf are remobilized to other parts of the plant. Whereas senescence is accompanied by a decline in leaf cytokinin content, exogenous application of cytokinins or an increase of the endogenous concentration delays senescence and causes nutrient mobilization. The finding that extracellular invertase and hexose transporters, as the functionally linked enzymes of an apolasmic phloem unloading pathway, are coinduced by cytokinins suggested that delay of senescence is mediated via an effect on source-sink relations. This hypothesis was further substantiated in this study by the finding that delay of senescence in transgenic tobacco (Nicotiana tabacum) plants with autoregulated cytokinin production correlates with an elevated extracellular invertase activity. The finding that the expression of an extracellular invertase under control of the senescence-induced SAG12 promoter results in a delay of senescence demonstrates that effect of cytokinins may be substituted by these metabolic enzymes. The observation that an increase in extracellular invertase is sufficient to delay leaf senescence was further verified by a complementing functional approach. Localized induction of an extracellular invertase under control of a chemically inducible promoter resulted in ectopic delay of senescence, resembling the naturally occurring green islands in autumn leaves. To establish a causal relationship between cytokinins and extracellular invertase for the delay of senescence, transgenic plants were generated that allowed inhibition of extracellular invertase in the presence of cytokinins. For this purpose, an invertase inhibitor was expressed under control of a cytokinin-inducible promoter. It has been shown that senescence is not any more delayed by cytokinin when the expression of the invertase inhibitor is elevated. This finding demonstrates that extracellular invertase is required for the delay of senescence by cytokinins and that it is a key element of the underlying molecular mechanism.     

Cytokinin biochemistry in relation to leaf senescence. VII. Endogenous cytokinin levels and exogenous applications of cytokinins in relation to sequential leaf senescence of tobacco

The cytokinin complex in tobacco leaves of various maturities was characterized by radioimmunoassay and mass spectrometry. Zeatin was the major base, whereas zeatin riboside was identified as the main riboside. in leaves of all maturities studied. Relative to upper younger leaves, the basal yellow leaves had reduced levels of both cytokinin bases and ribosides. Exogenous applications of dihydrozeatin and zeatin to detached tobacco leaves in amounts sufficient to delay senescence, elevated cytokinin base and riboside levels 2–5 fold. Presenescent and senescent leaves of intact plants showed quantitatively similar changes in cytokinin content. which therefore appear to be of significance in control of senescence. When supplied exogenously, the principal cytokinin bases found to occur in tobacco leaves (zeatin and dihydrozeatin) were markedly more effective than auxins and gibberellic acid in retarding senescence. Localised application of cytokinins to leaf blades of detopped plants was much less effective than application to intact plants. The cytokinin induced senescence retardation in tobacco leaves was independent of effects on directed metabolite transport. Evidence that endogenous levels of active cytokinins in intact tobacco leaves are involved in control of sequential leaf senescence is discussed.     

Phenotypically normal transgenic T-cyt tobacco plants as a model for the investigation of plant gene expression in response to phytohormonal stress

The tumour-inducing T-DNA gene 4 (T-cyt gene) of the nopaline Ti plasmid pTiC58 was cloned and introduced into tobacco cells by leaf disc transformation using Agrobacterium plasmid vectors. Tobacco shoots exposed to elevated cytokinin levels were unable to develop roots and lacked apical dominance. Using exogenously applied phytohormone manipulations we were able to regenerate morphologically normal transgenic tobacco plants which differed in endogenous cytokinin levels from normal untransformed plants. Although T-cyt gene mRNA levels, as revealed by dot-blot hybridization data, in these rooting plants were only about half those in primary transformed shoots the total amount of cytokinins was much lower than in crown gall tissue or cytokinin-type transformed shoots as reported by others. Nevertheless the cytokinin content in T-cyt plants was about 3 times greater than in control tobacco plants.Elevated cytokinin levels have been shown to change the expression of several plant genes, including some nuclear genes encoding chloroplast proteins. Our results show that the mRNA levels of chloroplast rbcL gene increase in cytokinin-type transgenic tobacco plants as compared with untransformed plants. Data obtained suggest that T-cyt transgenic plants are a good model for studying plant gene activity in different parts of the plant under endogenous cytokinin stress.     

The effect of plant cytokinin hormones on the production of ethylene, nitric oxide, and protein nitrotyrosine in ageing tobacco leaves

Transgenic plants with genetically increased or decreased levels of cytokinins were used to investigate the effect of cytokinin level on the production of ethylene, a plant hormone with suggested role in senescence, and the production of nitric oxide, potentially important signalling and regulatory molecule. The production of these gases was followed during the course of leaf development and senescence. The production of ethylene and nitric oxide is under genetic control of genes other than those involved in regulation of senescence. The difference in basic ethylene and NO levels in different tobacco cultivars was higher than their changes in senescence. The results of this study did not indicate a direct link between ethylene production and cytokinin levels. However, there was a decreased production of NO in senescent leaves. Low cytokinins level was associated with increased NO production during leaf development. Protein nitrotyrosine proved to be a better indicator of the reactive nitrogen species than measuring of the NO production. Higher nitrotyrosine concentrations were found in insoluble proteins than in the soluble ones, pointing to membrane proteins as the primary targets of the reactive nitrogen species. In plants with elevated cytokinin levels the content of nitrated proteins decreased both in soluble and insoluble fractions. This finding indicates an antioxidative function of cytokinins against reactive nitrogen species.     

Cytokinin biochemistry in relation to leaf senescence. VIII. Translocation, metabolism and biosynthesis of cytokinins in relation to sequential leaf senescence of tobacco

Cytokinin bases (zeatin and dihydrozeatin) and ribosides (zeatin riboside and dihydrozeatin riboside) were identified as major cytokinins in tobacco xylem sap by radioimmunoassay. When ³H-labelled zeatin riboside or dihydrozeatin riboside were supplied to tobacco plants via the xylem, leaves of differing maturity did not differ appreciably in level of radioactivity or in metabolism of the cytokinin. The major metabolites of zeatin riboside in leaves were adenine, adenosine and adenine nucleotides, whereas that of dihydrozeatin riboside was dihydrozeatin 7-glucoside. Incorporation of [¹⁴C]adenine into zeatin was evident in upper green leaves. indicating that young leaves have the capacity to synthesize cytokinins in situ. In contrast, fully expanded green leaves and senescing tobacco leaves exhibited little or no incorporation of [¹⁴C]adenine into cytokinins. This difference in cytokinin biosynthetic capacity may contribute to the differing cytokinin levels in leaves of different matirity, and may participate in control of sequential leaf senescence in tobacco.     

Ethylene and Cytokinin in the Control of Senescence in Detached Leaves of Arabidopsis thaliana eti-5Mutant and Wild-Type Plants

We studied the effects of cytokinin benzyladenine (BA) and ethylene on the senescence in the dark of detached leaves of Arabidopsis thaliana(L.) Heynh wild-type plants and theeti-5mutant, which was described in the literature as the ethylene-insensitive one. Leaf senescence was assessed from a decrease in the chlorophyll content. The content of endogenous cytokinins (zeatin and zeatin riboside) was estimated by the ELISA technique. We demonstrated that the content of endogenous cytokinins in the leaves of the three-week-old eti-5mutants exceeded that of the wild-type leaves by an order of magnitude; in the five-week-old mutants, by several times; and in the seven-week-old plants, the difference became insignificant. Due to the excess of endogenous cytokinins in the three–five-week-old mutant leaves, their senescence in the dark was retarded and exogenous cytokinin affected these leaves to a lesser extent. The seven-week-old mutant and the wild-type leaves, which contained practically similar amounts of endogenous cytokinins, did not differ in these indices. Thus, the level of endogenous cytokinins determined the rate of senescence and the leaf response to cytokinin treatment. Ethylene accelerated the senescence of detached wild-type leaves. Ethylene action increased with increasing its concentration from 0.1 to 100 l/l. BA (10^–6M) suppressed ethylene action. Similar data were obtained for the eti-5mutant leaves. We therefore suggest that the mutant leaves comprised the pathways of the ethylene signal reception and transduction, which provided for the acceleration of their senescence.     

Cytokinins and cytokinin-binding sites in transgenic potato plants

The introduction of the gene for cytokinin biosynthesis into the potato genome led to a manifold increase in the level of cytokinins (zeatin and zeatin riboside) in transgenic plants grown in vitro. The high amount of endogenous cytokinins in 20-day-old plants of clone 1339-3A correlated with high cytokinin-binding capacity of ribosomes that is presumably attribute to a cytokinin receptor.     

Expression of Phospholipase D during Castor Bean Leaf Senescence

Membrane deterioration in plant senescence is commonly associated with progressive decreases in membrane phospholipid content. This study investigated the expression and regulation of phospholipase D (PLD; EC 3.1.4.4) during senescence in castor bean (Ricinus communis L. cv Hale) leaf discs. The rate of leaf senescence was accelerated by 50 [mu]M abscisic acid and was attenuated by 50 [mu]M cytokinin during incubation at 23[deg]C for up to 5 d. Leaf senescence was indicated by decreases in the content of total proteins, chlorophyll, and phospholipids. PLD activity in both membrane-associated and cytosolic fractions showed a gradual increase in the absence of phytohormones. Abscisic acid stimulated an increase in membrane-associated PLD and had little effect on the soluble form. On the other hand, cytokinin retarded the increase in membrane-associated PLD. Immunoblotting analysis using PLD-specific antibodies revealed that the changes in PLD activity were correlated with those of PLD protein. Analysis of PLD by nondenaturing PAGE showed the appearance of a PLD structural variant, PLD 3, in abscisic acid-treated leaf discs. Northern blotting analysis using a PLD cDNA probe revealed an increase in PLD mRNA in senescing leaf discs. These data indicate complex mechanisms for the regulation of PLD during senescence, which include increases in membrane-associated PLD, differential expression of PLD isoforms, and changes in amounts of PLD protein and mRNA. Such controlled expression points to a role for PLD in membrane deterioration and plant senescence.     

Effects of early-fruit removal on endogenous cytokinins and abscisic acid in relation to leaf senescence in cotton

Numerous studies have shown that early-fruit removal enhances vegetative growth and development of cotton (Gossypium hirsutum L.). However, few studies have examined changes in leaf senescence and endogenous hormones due to fruit removal. The objective of this study was to determine the correlation between some endogenous phytohormones, particularly the cytokinins and abscisic acid (ABA), and leaf senescence following fruit removal. Cotton was grown in pots and in the field during 2005 and 2006. Two early-fruiting branches were excised from plants at squaring to form the fruit removal treatment while the non-excised plants served as control. Plant biomass, seed cotton yield, cytokinins and ABA levels in main-stem leaves and xylem sap as well as main-stem leaf photosynthetic rate (Pn) and chlorophyll (Chl) concentration were determined after removal or at harvest. Fruit removals increased the leaf area, root and shoot dry weight and plant biomass at 35 days after removal (DAR), whether in potted or field-grown cotton; under field conditions, it also improved plant biomass and seed cotton yield at harvest. The Pn and Chl concentration in excised plants were significantly higher than in control plants from 5 to 35 DAR, suggesting that fruit removal considerably delayed leaf senescence. Fruit-excised plants contained more trans-zeatin and its riboside (t-Z + t-ZR), dihydrozeatin and its riboside (DHZ + DHZR), and isopentenyladenine and its riboside (iP + iPA) but less ABA in both main-stem leaves and xylem sap than control plants from 5 to 35 DAR. These results suggest that removal of early fruiting branches delays main-stem leaf senescence, which can be attributed to increased cytokinin and/or reduced ABA. Cytokinin and ABA are involved in leaf senescence following early fruit removal.     

Senescence-Inducible Expression of Isopentenyl Transferase Extends Leaf Life, Increases Drought Stress Resistance and Alters Cytokinin Metabolism in Cassava

Cassava (Manihot esculenta Crantz) sheds its leaves during growth, especially within the tropical dry season. With the production of SAG12-IPT transgenic cassava we want to test the level of leaf retention and altered cytokinin metabolism of transgenic plants via the autoregulatory senescence inhibition system. After confirmation of transgene expression by molecular analysis and phenotype examination in greenhouse plants, two transgenic plant lines, 529-28 and 529-48, were chosen for further investigation. Detached mature leaves of 529-28 plants retained high levels of chlorophyll compared with wild-type leaves after dark-induced senescence treatment. Line 529-28 showed significant drought tolerance as indicated by stay-green capacity after drought stress treatment. Field experiments proved that leaf senescence syndrome was significantly delayed in 529-28 plants in comparison with wild-type and 529-48 plants. Physiological and agronomical characterizations of these plants also revealed that the induced expression of IPT had effects on photosynthesis, sugar allocation and nitrogen partitioning. Importantly, the 529-28 plants accumulated a high level of trans-zeatin-type cytokinins particularly of corresponding storage O-glucosides to maintain cytokinin homeostasis. Our study proves the feasibility of prolonging the leaf life of woody cassava and also sheds light on the control of cytokinin homeostasis in cassava leaves.     

Cytokinin Activity in Lupinus albus

The cytokinin content of the root exudate and leaves of fruiting white lupin plants (Lupinus albus L.) was investigated at 2 weekly intervals after anthesis of the lowest flower on the primary inflorescence. Up to 8 weeks after anthesis the level of cytokinins in the root exudate increased. However, at 10 weeks after anthesis insufficient sap was produced for analysis. Cytokinins co-eluting with zeatin and zeatin riboside were detected in the root exudate after fractionation on Sephadex LH-20. The cytokinin levels in the mature leaves steadily increased up to 8 weeks after anthesis and thereafter remained relatively constant. Three peaks of activity, co-eluting with zeatin, zeatin riboside and the glucoside cytokinins were recorded in the leaf extracts. The level of glucoside cytokinins in the leaves was high at 8 and 10 weeks after anthesis. Paper chromatography of extracts of fruits collected at 2 weeks after anthesis indicated that as fruit development proceeded there was a build up of cytokinin in this region of the plant. It is suggested that, in the white lupin, the cytokinins translocated to the shoot are accumulated in the leaves and in the fruits and that it is only later when there is a considerable decrease in sap (10 weeks after anthesis) production that a decreasing supply of cytokinins leads to shoot senescence.     

Contrasting patterns of cytokinins between years in senescing aspen leaves

Cytokinins are plant hormones that typically block or delay leaf senescence. We profiled 34 different cytokinins/cytokinin metabolites (including precursors, conjugates and degradation products) in leaves of a free‐growing mature aspen (Populus tremula) before and after the initiation of autumnal senescence over three consecutive years. The levels and profiles of individual cytokinin species, or classes/groups, varied greatly between years, despite the fact that the onset of autumn senescence was at the same time each year, and senescence was not associated with depletion of either active or total cytokinin levels. Levels of aromatic cytokinins (topolins) were low and changed little over the autumn period. Diurnal variations and weather‐dependent variations in cytokinin content were relatively limited. We also followed the expression patterns of all aspen genes implicated as having roles in cytokinin metabolism or signalling, but neither the pattern of regulation of any group of genes nor the expression of any particular gene supported the notion that decreased cytokinin signalling could explain the onset of senescence. Based on the results from this tree, we therefore suggest that cytokinin depletion is unlikely to explain the onset of autumn leaf senescence in aspen.     

AtMYB2 regulates whole plant senescence by inhibiting cytokinin-mediated branching at late stages of development in Arabidopsis

Whole plant senescence of monocarpic plants consists of three major processes: arrest of shoot apical meristem, organ senescence, and permanent suppression of axillary buds. At early stages of development, axillary buds are inhibited by shoot apex-produced auxin, a mechanism known as apical dominance. How the buds are suppressed as an essential part of whole plant senescence, especially when the shoot apexes are senescent, is not clear. Here, we report an AtMYB2-regulated post apical dominance mechanism by which Arabidopsis (Arabidopsis thaliana) inhibits the outgrowth of axillary buds as part of the whole plant senescence program. AtMYB2 is expressed in the compressed basal internode region of Arabidopsis at late stages of development to suppress the production of cytokinins, the group of hormones that are required for axillary bud outgrowth. atmyb2 T-DNA insertion lines have enhanced expression of cytokinin-synthesizing isopentenyltransferases genes, contain higher levels of cytokinins, and display a bushy phenotype at late stages of development. As a result of the continuous generation of new shoots, atmyb2 plants have a prolonged life span. The AtMYB2 promoter-directed cytokinin oxidase 1 gene in the T-DNA insertion lines reduces the endogenous cytokinin levels and restores the bushy phenotype to the wild type.