The Relationship of Cytokinins and Senescence A Manipulation of Cytokinin Levels

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Cytokinins were originally discovered as factors promoting plant cell division (Miller et al., 1956). Since this discovery, cytokinins have been shown to have effects on many different aspects of plant development. For example, cytokinin suppresses apical dominance and promotes the growth of lateral buds (Phillips, 1975) and viviparous leaves (Estruch et al., 1991), chloroplast development (Parthier, 1979), leaf expansion (Miller et al., 1956), and delays tissue senescence (Richmond and Lang, 1957; Van Staden etal., 1988). The extensive literature on the role of cytokinins has been reviewed elsewhere (e.g., Binns, 1994; Mok and Mok, 1994), and this section will focus on the role of cytokinins in senescence and the genetic engineering efforts to delay senescence by modulating endogenous cytokinin levels.

The first evidence that cytokinin can delay senescence was from the study of exoge-nously applied cytokinin (Richmond and Lang, 1957). Subsequently an extensive literature has developed describing general antagonistic effects of applied cytokinin on senescence (reviewed in Van Staden et al., 1988). The discovery of an inverse correlation between cytokinin levels and the progression of senescence (e.g., Nooden andLetham, 1993; Ambler et al., 1992; Soejima et al., 1995) provided further evidence that endogenously produced cytokinin has a negative effect on plant senescence. Relatively recent approaches using transgenic plants expressing the gene encoding isopentenyl transferase (IPT) from the T-DNAof Agrobacteriumtumefaciens (Akiyoshi etal., 1983; Barry etal., 1984) have provided a tool to manipulate endogenous cytokinin levels and to study the role of cytokinin in plant development. The transgenic plant approach has the advantage of precisely manipulating both spatial and temporal levels of cytokinin (Klee, 1994). One of the consistent results obtained from studies using transgenic plants with altered cytokinin biosynthesis is that senescence is delayed in plant tissues in which cytokinin levels are elevated.

A variety of expression systems have been used to modulate endogenous cytokinin levels. Generally, plant tissues transformed with IPT under the control of constitutive promoters spontaneously produce shoots, but the transformed shoots cannot form roots as expected for cytokinin overproducing cells (Schmulling et al., 1989; Binns, 1994). Therefore, when plants containing constitutively expressed IPT are regenerated, there has probably been selection for weak expression, particularly in roots (Gan and Amasino, 1996). Many different approaches have been undertaken to avoid this problem.

One approach is based upon inducible promoter systems to provide for conditional IPT expression. Heatshock promoters have often been used. Smart et al. (1991) reported that transgenic tobacco plants transformed with IPT under the control of a soybean heatshock promoter showed phenotypes associated with higher cytokinin levels such as shorter stature, lateral shoot growth, and delayed leaf senescence even without heatshocks. These pheno-types were more profound after several heatshocks of whole plants or defined areas of leaves. However, Medford et al. (1989) did not observe a phenotypic difference before and after heatshocks in transgenic tobacco and Arabidopsis plants transformed with IPT under the control of a maize heatshock promoter. With or without heatshock, the transgenic plants exhibited reduced apical dominance and a delay in leaf senescence. As expected from the phenotypic change in non-heatshocked plants, cytokinin levels in transgenic plants were higher than control plants even in the absence of heatshock, but after heatshock cytokinin levels were further elevated. Other studies using heat-inducible promoters also revealed leaky expression of IPT without heatshock (Schmulling et al., 1989; Smigocki, 1991; Ainley et al., 1993), and the low level of IPT activity driven by this leaky expression is usually enough to cause cytokinin overproduction phenotypes. The elevated cytokinin levels caused by the leaky expression may be above a threshold such that additional cytokinin produced by heatshock does not result in stronger phenotypes (Binns, 1994). A concern with heat-inducible promoter systems is that heatshock could itself affect plant responses. Other inducible promoters responding to wounding (Smigocki et al., 1993) and light (Beinsberger et al., 1992) have also been used to direct IPT expression. More recently, tetracycline-(Faiss et al., 1997) and copper-inducible (McKenzie et al., 1998) systems have been used to direct IPT expression in transgenic tobacco plants. Generally, these systems have tighter control compared to heat-inducible systems, although delayed senescence was observed from a transgenic tobacco line harboring the tetracycline-inducible system even without tetracycline treatment. In the case of transgenic tobacco plants harboring the copper-inducible system, a copper-treatment-dependent delay in leaf senescence was observed. Perhaps the most significant limitation of the inducible systems is that the delayed tissue senescence obtained from transgenic plants expressing IPT under the control of inducible promoters is always associated with other abnormal phenotypes resulting from cytokinin overproduction.

A second approach is based on tissue- or developmental-specific promoter systems. In theory, these systems could result in more specific overproduction of cytokinin and minimized abnormal phenotypes in undesired tissues or developmental stages. When an auxin-inducible SAUR promoter was used to drive IPT expression, numerous phenotypes commonly associated with cytokinin overproduction were observed in transgenic tobacco plants (Li et al., 1992). Delayed leaf senescence was observed when these plants were grown in a growth chamber, while accelerated leaf senescence was observed when the same plants were grown in tissue-culture vessels. Different growth conditions might have resulted in different translocation patterns of cytokinins. The SAUR promoter is most active in elongating regions of transgenic tobacco hypocotyls and stems. Active transpiration in growth chamber-grown plants might have resulted in sufficient translocation of cytokinins to leaves resulting in delayed senescence, while lack of transpiration in culture-grown plants might account for the different phenotype (Li et al., 1992). Martineau et al. (1994) used a fruit-specific promoter to drive the expression of IPT in tomato, and observed an altered fruit phenotype: islands of green pericarp tissue remaining on otherwise red ripe fruit. Cytokinin levels in these transgenic tomato fruit were 10- to 100-fold higher than in control fruit. A four-fold increase in cytokinin levels in the leaves of transgenic tomatoes was also observed despite a lack of detectable IPT mRNA accumulation in this tissue.

To specifically provide cytokinin to senescing leaves, a senescence-delaying system has been developed. When the expression of IPT was controlled by the promoter of an Arabidopsis senescence-specific gene, SAG12, transgenic tobacco plants containing this expression cassette exhibited a strong delay in leaf senescence (Gan and Amasino, 1995; Fig. 6-1). This senescence-delaying system is autoregulatory: at the onset of senescence the senescence-specific promoter is activated resulting in cytokinin production, and the increase in cytokinin will in turn inhibit senescence and attenuate the promoter activity. This system has three important features: spatially, cytokinin production is targeted to tissues where the promoter is active, i.e. to leaves and floral parts; temporally, cytokinin production is controlled developmentally, i.e. cytokinins are produced only during senescence; and, quantitatively, cytokinins are maintained at a minimum level necessary for the inhibition of senescence owing to autoregulation (Gan and Amasino, 1996). Transgenic tobacco plants

Senescence-specific promoter



t 1

Isopantenyl transferase

Figure 6-1. Autoregulated senescence-inhibition system. (A) Schematic of the system. Senescence is inhibited through the autoregulation between the senescence-specific production of cytokinins and the suppression of the senescence-specific promoter by the produced cytokinins. (B) Delayed leaf senescence in a transgenic tobacco plant containing the autoregulated senescence-inhibition system. Left, transgenic plant; right, wild-type. (Modified from Gan and Amasino, 1996.)

Figure 6-1. Autoregulated senescence-inhibition system. (A) Schematic of the system. Senescence is inhibited through the autoregulation between the senescence-specific production of cytokinins and the suppression of the senescence-specific promoter by the produced cytokinins. (B) Delayed leaf senescence in a transgenic tobacco plant containing the autoregulated senescence-inhibition system. Left, transgenic plant; right, wild-type. (Modified from Gan and Amasino, 1996.)

containing this expression system are developmentally and morphologically normal except for the significant delay in leaf senescence. These transgenic plants also show an increase in seed yield and biomass, probably because of the prolonged photosynthetic activity of senescence-retarded leaves. Because the cytokinin production is controlled genetically and developmentally in this system, no special treatments or growth conditions are required to obtain delayed senescence and increased yield.

It is notable that overexpression of certain genes other than IPT also causes increased cytokinin levels. Expression of a gene encoding a rice small GTP-binding protein in tobacco caused six-fold higher cytokinin levels and phenotypes associated with cytokinin overproduction (Kamada et al, 1992; Sano et al, 1994). Higher cytokinin levels and associated phenotypes were also observed in transgenic tobacco plants overexpressing either a tobacco gene, NTH15 (Tamaoki et al., 1997), or a rice gene, OSH1 (Kusaba et al., 1998). Proteins encoded by these genes belong to the Knotted class of homeodomain proteins. Thus this class of homeodomain proteins may regulate cytokinin synthesis. Moreover, when the maize Knotted1 gene was expressed under the control of a senescence-specific promoter in tobacco, the resulting transgenic plants showed increased cytokinin levels in senescing leaves and delayed leaf senescence (Ori et al., 1999). Therefore, several reports demonstrate that genetic manipulation of endogenous cytokinin levels can be achieved by the altered expression of certain plant genes other than IPT. Transgenic plant systems that have resulted in altered endogenous cytokinin levels are summarized in Table 6-1.

B. Cytokinin Signaling and Mechanism of Action

The diverse effects of cytokinin on plant development suggest that the expression of a variety of genes are affected by this hormone, and indeed many classes of genes including those involved in plant defense responses, photosynthesis, and senescence have been shown to be regulated by cytokinin. Cytokinin signaling pathways leading to this gene expression are not yet fully understood, although significant progress has been made recently.

Early studies on cytokinin regulation of gene expression were performed in cytokinin-dependent soybean cell suspension cultures. Polyribosome formation and new protein synthesis were observed after cytokinin treatment (Fosket and Tepfer, 1978) and 20 mRNAs, including some of ribosomal proteins, that exhibit rapid accumulation following cytokinin treatment were identified (Crowell et al., 1990). These studies indicated that translation and transcription of many genes are affected by cytokinin treatment. However, many of the genes induced by cytokinin were also induced by auxin, indicating that their induction may be specific to growth stimulation rather than to a particular hormone (Binns, 1994).

Many genes thought to be involved in defense responses show altered expression in response to cytokinin. j-1,3-Glucanase, chitinase, and some pathogenesis-related (PR) genes exhibit decreased expression in response to increased cytokinin (Eichholz et al., 1983; Mohnen et al., 1985; Felix and Meins, 1986; Shinshi et al., 1987). In other reports, chitinase and PR genes exhibited increased expression in cells with increased cytokinin levels (Memelink et al., 1987; Simmons et al., 1992; Martineau et al., 1994). Sano et al. (1994) demonstrated that transgenic tobacco plants expressing rgp1, a gene encoding a Ras-related small GTP-binding protein, exhibit increased cytokinin levels, high salicylic acid levels, increased acidic PR gene expression, and a high level of resistance against tobacco mosaic virus infection.

Table 6-1. a Transgenic Plant Systems that have Resulted in Altered Endogenous Cytokinin Levels


Source of promoter

Transgenic species


IPT systems

IPT native promoter

Constitutive expression

Transposition Random insertion Inducible Heat

Wounding Light

Tetracycline Copper Tissue-specific Elongating region Fruit-specific Ovary-preferential Development-specific Senescence-specific Non-IPT systems Homeobox protein NTH15 OSH1

Knotted1 Small GTP binding protein

From pTi15955

From pTiC58 From pTi15955

CaMV 35S

CaMV 35S promoter disrupted by Ac transposon Unknown

Maize hsp70

Drosophila hsp70

Soybean HS6871

Soybean Gmhsp17.5-E

Potato proteinase inhibitor II-K gene

Pea rubisco small subunit gene

Tetracycline-dependent CaMV 35S

Yeast copper-metallothionein regulatory system

Soybean SAUR gene Tomato 2A11 gene Tomato unknown gene

Arabidopsis SAG12


Arabidopsis SAG12 CaMV 35S

Nicotiana tabacum Solanum tuberosum N. tabacum N. tabacum

Lycopersicon esculentum N. tabacum N. plumbaginifolia Cucumis sativa N. tabacum N. tabacum

N. tabacum Arabidopsis thaliana N. tabacum N. plumbaginifolia N. tabacum N. tabacum N. plumbaginifolia N. tabacum N. tabacum N. tabacum

N. tabacum L. esculentum L. esculentum

N. tabacum

N. tabacum N. tabacum N. tabacum N. tabacum N. tabacum

Schm├╝lling etal., 1989 Ooms etal., 1991

Yushibov etal., 1991; Beinsberger etal., 1992 Zhang et al., 1995 Groot etal., 1995 Smigocki and Owens, 1988

Estruch etal., 1991, 1993 Hewelt etal., 1994

Medford etal, 1989

Schm├╝lling etal., 1989 Smigocki, 1991 Smart etal., 1991 Ainley etal., 1993 Smigocki etal., 1993 Beinsberger et al., 1992 Faiss etal., 1997 McKenzie etal., 1998

Li etal., 1992 Martineau etal., 1994 Martineau etal., 1994

Gan and Amasino, 1995

Tamaoki et al., 1997 Kusaba et al., 1998

Ori etal., 1999 Sano etal., 1994

a Updated from Gan and Amasino (1996).

Because photosynthetic activity declines during plant senescence (Gepstein, 1988; Hensel et al, 1993; Jiang et al, 1993), the effect of cytokinin on photosynthesis is of particular interest. Consistent with the function of cytokinin in chloroplast development, genes involved in photosynthesis are up regulated by cytokinin treatment (reviewed in Crowell and Amasino, 1994). Cytokinin is known to induce the expression of chlorophyll a/b binding protein (CAB) and ribulose-1,5-bisphosphate small and large subunit mRNAs in many different plant species, while the decrease of CAB mRNA and chlorophyll content in senescing Arabidopsis leaves is delayed by cytokinin treatment (Weaver et al., 1998). Light-dependent activation of a wheat protein kinase gene, wpk4, was found to be dependent on cytokinin (Sano and Youssefian, 1994), suggesting a role of cytokinin in mediating light-dependent activation of regulatory genes.

During the past several years, a number genes showing increased or specific expression during senescence (senescence-associated genes or SAGs) have been isolated from a variety of plant species (Davies and Grierson, 1989; Azumi and Watanabe, 1991; Becker and Apel, 1993; Hensel et al, 1993; Taylor et al., 1993; Buchanan-Wollaston, 1994; Lohman et al., 1994; King etal, 1995; Smart etal., 1995; Drake etal, 1996;Hanfrey etal., 1996; Oh etal., 1996; Buchanan-Wollaston and Ainsworth, 1997; Park et al, 1998; Weaver et al, 1998; Quirino et al., 1999; Noh and Amasino, 1999a,b; Chapter 4). Studies on the expression pattern of SAGs have revealed that induction of most SAG mRNAs is delayed or repressed by cytokinin treatment (Weaver et al., 1998; Noh and Amasino, 1999a,b). Because the function of most SAGs during senescence is not yet clear, it is not known whether the decreased expression of SAGs is the cause or the result of the delayed senescence after cytokinin treatment. Elucidation of the function of individual SAGs during senescence will help to reveal the mechanisms of cytokinin action in delaying plant senescence.

Difficulties in obtaining mutants having altered cytokinin activity or responsiveness have hindered the application of genetics to understanding cytokinin metabolism and signal transduction. However, several recent reports have implicated two types of signaling pathway components in cytokinin action: a G-protein-coupled receptor and a two-component histidine kinase (Estelle, 1998). Reduced cytokinin sensitivity has been observed from transgenic Arabidopsis constitutively expressing antisense mRNA of GCR1, a G-protein-coupled receptor homologue (Plakidou-Dymock et al., 1998). Kakimoto (1996) demonstrated that ectopic expression of CKI1, a homologue of two-component histidine kinases that consist of a sensor and a response regulator, results in cytokinin-independent cell division in cultured Arabidopsis tissues. Moreover, Brandstatter and Kieber (1998) demonstrated that IBC6, a member of the response regulator group, exhibits rapid cytokinin-specific induction of its mRNA. Considering the broad effects of cytokinin on plant development, downstream cytokinin signaling pathways are likely to be branched and complex. Alterations in cytokinin perception or early signaling events, especially in a tissue- or developmental-specific manner, have much potential for crop-improving efforts.

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