Research Paper Abstract: Two promoters of senescence-associated Arabidopsis genes, SAG12 and SAG13, were used in tomato plants to express IPT that catalyzes the rate-limiting step in cytokinin biosynthe- sis. Expression of these heterologous promoters in tomato plants was analyzed using the reporter gene β-glucuronidase. Both promoters are expressed in tomato leaves in a manner sim- ilar to their expression in Arabidopsis plants. The SAG12 promoter is very specific to senescing leaves, whereas the SAG13 promoter is expressed in mature leaves prior to the onset of visible senes- cence and its expression increases in senescing leaves. Expres- sion of both promoters in tomato tissues other than leaves was very low. IPT expressed under the control of SAG12 and SAG13 promoters (P SAG12 ::IPT and P SAG13 ::IPT, respectively) resulted in suppression of leaf senescence and advanced flowering, as well as in a slight increase in fruit weight and fruit total soluble solids (TSS). However, expression of P SAG13 ::IPT also led to stem thicken- ing, short internodal distances and loss of apical dominance. In contrast to the autoregulation of P SAG12 ::IPT, P SAG13 ::IPT is ex- pressed at higher levels in mature leaves. This difference is likely due to P SAG13 ::IPT exhibiting two phases of expression – a senes- cence-independent expression prior to the onset of senescence that is not subjected to autoregulation by cytokinin, and en- hanced expression throughout senescence which is autoregualt- ed by cytokinin. This moderate different autoregulated behavior of P SAG12 ::IPT and P SAG13 ::IPT markedly influenced plant develop- ment, emphasizing the biological effects of cytokinin in addition to senescence inhibition. Key words: Cytokinin, IPT, promoter, SAG, senescence, tomato. Abbreviations: SAG: senescence-associated gene TSS: total soluble solids IPT: isopentyl transferase GUS: β-glucuronidase Introduction Leaf senescence is an active orderly process characterized by a reduced rate of photosynthesis, loss of chlorophyll, leaf yel- lowing, degradation of proteins, and recycling of nutrients to young tissues (Gan and Amasino, 1997; Smart, 1994). Leaf se- nescence is regulated by the combined action of external and internal factors such as light intensity, nutrient availability, leaf age, and reproductive stage (Gan and Amasino, 1997; Smart, 1994). Plant hormones such as auxins, gibberellins, ethylene, abscisic acid, and cytokinins are also involved in the regulation of senescence. Cytokinins, which are thought to be synthesized mainly in the roots and transported to the shoots via the xylem (Gan and Amasino, 1996), have been implicated in several aspects of plant development and are key compo- nents in the regulation of plant senescence (Buchanan-Wollas- ton,1997; Gan and Amasino, 1997; Nam, 1997; Gan and Ama- sino, 1996; Santokh et al., 1992a, b; Van-Staden et al., 1988). Endogenous cytokinin concentrations decrease in senescing plant tissues (Nooden et al., 1990; Van-Staden et al., 1988; Skene, 1975) and exogenous application of cytokinin retards senescence (Gan and Amasino, 1996). Transgene-encoded cytokinin biosynthesis was initially stud- ied in tobacco (Nicotiana tabacum) using constitutive or induc- ible over-expression of the IPT gene from Agrobacterium tume- faciens, which encodes isopentenyl transferase. This enzyme catalyzes the rate-limiting step for the addition of dimethyl- allyl to the N6 of 5-AMP to form isopentenyl AMP, which is the precursor of all other cytokinins (Chen, 1997; McGaw and Burch, 1995). Over-expression of the IPT gene in transgenic plants led to elevated foliar cytokinin concentrations and de- layed leaf senescence. However, the high cytokinin levels were largely detrimental to growth and fertility (McKenzie et al., 1998; Wang et al.,1997; Hewelt et al., 1994; Van Loven et al., 1993; Li et al., 1992; Smart et al., 1991; Smigocki,1991; Med- ford et al., 1989). Gan and Amasino (1995) devised autoregu- lated cytokinin production that delayed leaf senescence in transgenic tobacco plants without altering the plant pheno- type. This strategy exploited a highly senescence-specific pro- moter (P SAG12 ) from an Arabidopsis gene SAG12 encoding a cys- teine protease (Lohman et al., 1994) fused to the IPT gene from the Ti plasmid of A. tumefaciens. The fused P SAG12 ::IPT gene was activated only at the onset of senescence in the lower mature tobacco leaves (Gan and Amasino, 1995). This resulted in cyto- kinin biosynthesis in the leaves, which inhibited leaf senes- Effects of Cytokinin Production under Two SAG Promoters on Senescence and Development of Tomato Plants D. Swartzberg 1 , N. Dai 1 , S. Gan 2 , R. Amasino 3 , and D. Granot 1 1 Institute of Plant Sciences, Agricultural Research Organization, The Volcani Center, P.O. Box 6, Bet Dagan, 50250, Israel 2 Department of Horticulture, Cornell University, Ithaca, NY 14853-5904, USA 3 University of Wisconsin, 433 Babcock Drive, Madison, WI 53706, USA Received: February 7, 2006; Accepted: April 26, 2006 Plant Biol. © Georg Thieme Verlag KG Stuttgart · New York DOI 10.1055/s-2006-924240 · Published online 2006 ISSN 1435-8603 1