1. LEAF SENESCENCE, LEVELS OF CYTOKININS AND NITRATE AND ACTIVITY OF THEIR DEGRADATION ENZYMES IN WILD TYPE AND TRANSGENIC P SAG 12 -IPT WHEAT CV. SCAMP PLANTS. 1 Blanka ŠOLCOVÁ, 2 Gabriela JANDOVÁ, 1 Václav MOTYKA, 2 Marie TRČKOVÁ, 3 Malcolm C. ELLIOTT, 1 Miroslav KAMÍNEK 1 Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Rozvojová 135, Prague 6, 165 02, Czech Republic, phone: +42020390436, fax: +42020390446, E- mail: solcova@ueb.cas.czsolcova@ueb.cas.cz 2 Research Institute of Crop Production, Drnovská 505, Prague 6, 161 00, Czech Republic 3 The Norman Borlaug Institute for Plant Science Research, De Montfort University, Scraptoft, Leicester LE7 9SU, U.K. Material and Methods Plant material: Wheat (Triticum aestivum L., cv. Scamp) wild type and the ipt transformed plants with senescence inducible promotor (P SAG 12 – IPT). Cultivation: Both P SAg 12 –IPT transgenic and wild type wheat were cultivated in hydroponic conditions (82,5  M MgSO 4, 190  M KH 2 PO 4, 105  M KCl) at 16 / 8 h and 20 / 15°C (day / night) under optimum (1158  M) and suboptimum (773  M) NO 3 - supply. Transformation: The wheat immature embryos were co-bombarded by plasmids pSG516 and pDB1. Plasmid pSG516 contained PSAG 12-ipt as the target construct while plasmid pDB1 contained gusA and bar as reporter and selectable marker, respectively. Selection of transformants was performed on MS medium without plant growth regulators but supplemented with 5 mg/l bialaphos. Presence of ipt in DNA was determined using PCR. Senescence progress was detected on the chlorophyll loss basis. Chlorophyll was extracted using 85% acetone acording to AOAC Official Methods of Analysis. Endogenous cytokinins were isolated and quantified using HPLC/MS according to Dobrev and Kaminek (2002). Nitrate content was estimated spectrophotometrically using a Skalar San plus analyzer. Nitrate uptake was determined by depletion from nutrient solution. Cytokinin oxidase/dehydrogenase activity was estimated by in vitro conversion of [2,8- 3 H] isopentenyladenine to [2,8- 3 H] adenine according to Motyka et al (1996). Nitrate reductase activity was determined by an in vitro assay using azo dye. Introduction Leaf senescence is an integral part of plant development. It is accelerated by different external stimuli including mineral deficiency, especially deficiency of nitrogen. As programmed process it is under control of internal mechanisms which include hormonal regulations. Cytokinins (CKs) are known to slow-down the progress of senescence. Expression of most genes is down-regulated during the progressing leaf senescence but expression of distinct sets of genes, so called SAGs (senescence-associated genes), is increased (for review see Yoshida 2003). We compare here the progress of senescence, changes in CK levels and in uptake and assimilation of nitrogen in transgenic wheat plants transformed with P SAG 12 -IPT and untransformed controls. Results Chlorophyll content decreased with the leaf insertion and age and the senescence was delayed in leaves of transgenic P SAG 12 –IPT plants as compared to control ones (fig. 1). Optimal nitrate level in medium a line the differences in chlorophyll content in flag leaf between controls and ipt transformed plants. The physiologically active CKs in flag leaves of transgenic P SAG 12 –IPT plants were to large extent converted to storage O-glucosides and inactive N-glucosides. Transgenic P SAG 12 –IPT plants also contained much higher amount of inactive derivates of cis-zeatin (table 1) High level of active cytokinin forms remain in control plants. Optimal nitrate level shift cytokinins in wild type plants towards to inactive N-glucosides. Cytokinin oxidase/dehydrogenase (CKX) was more active in transgenic P SAG 12 –IPT plants compared to controls but the optimal nitrate level decreased the CKX activity (table 2). The nitrate reductase (NR) activity was higher in flag leaves than in older leaves in both control and ipt transformed plants (fig. 2). Leaves of transgenic P SAG 12 -IPT plants displayed significantly lower NR activity than leaves of corresponding controls and levels of nitrate positively correlated with NR activity (fig. 1). These results indicate that accumulation of active CKs in transgenic P SAG 12 -IPT plants is accompanied with their inactivation and degradation to maintain cytokinin homeostasis. Lowering of nitrate levels and activity of NR in leaves of transgenic P SAG 12 –IPT plants implies that nitrate uptake was either decreased or its assimilation was enhanced in ipt transformed plants. CYTOKININ CONTENT IN WILD TYPE AND TRANSGENIC P SAG 12 -IPT WHEAT CV. SCAMP [pmol / g FW] Table 1: control = wild-type wheat, P SAG 12 –IPT= ipt transformed plants with senescence inducible promotor, B+R = bases and ribosides (cis and trans zeatin, isopentenyladenine and dihydrozeatin and their ribosides), O-G = O-glucosides of zeatin, zeatin riboside and dihydrozeatin, N-G = N 7 - and N 9 –glucosides of zeatin, dihydrozeatin and isopentenyladenine, ribotides – of trans and cis zeatin riboside, dihydrozeatin riboside and isopentenyladenine riboside, cis derivates of zeatin N 7 - and N 9 –glucosides, zeatin O-glucoside and zeatine riboside O-glucoside, Σ = total cytokinin content, LN (low nitrate) = suboptimal level of NO 3 - (773  M) in medium, HN (high nitrate) = optimal level of NO 3 - (1158  M) in medium. Nitrate levelB+RO-GN-Gribotides cis derivates Σ controlLN 142,819,894,011,6820,91089,1 HN 3,433,2129,110,51520,41696,6 P SAG 12 -IPTLN 10,239,132,112,01602,51695,9 HN 6,515,98,38,91370,01409,6 NITRATEREDUCTASE ACTIVITY IN WILD TYPE AND TRANSGENIC P SAG 12 -IPT WHEAT CV. SCAMP 1 st leaf 2 nd leaf Leaf position controlP SAG 12 -IPT LNHN Nitrate reductase activity [nmol NO 3 - / g FW / min] Figure 3: control = wild type wheat, P SAG 12 – IPT = ipt transformed plants with senescence inducible promotor, 1 st leaf = flag leaf, LN (low nitrate) = suboptimal level of NO 3 - (773  M) in medium, HN (high nitrate) = optimal level of NO 3 - (1158  M) in medium. Columns characterize nitrate reductase activity in nmol NO 3 - / g leaf fresh weight / min. CHLOROPHYLL CONTENT IN WILD TYPE AND TRANSGENIC P SAG 12 -IPT WHEAT CV. SCAMP 1 st leaf 2 nd leaf Leaf position Chlorophyll content [mg / g FW] controlP SAG 12 -IPT LNHN Figure 1: control = wild type wheat, P SAG 12 – IPT = ipt transformed plants with senescence inducible promotor, 1 st leaf = flag leaf, LN (low nitrate) = suboptimal level of NO 3 - (773  M) in medium, HN (high nitrate) = optimal level of NO 3 - (1158  M) in medium. Columns characterize the chlorophyll (a+b) content in mg / g leaf fresh weight. NITRATE CONTENT IN WILD TYPE AND TRANSGENIC P SAG 12 -IPT WHEAT CV. SCAMP 1 st leaf 2 nd leaf Leaf position Nitrate content [  mol NO 3 - / g FW] LNHN controlP SAG 12 -IPT Figure 2: control = wild type wheat, P SAG 12 – IPT = ipt transformed plants with senescence inducible promotor, 1 st leaf = flag leaf, LN (low nitrate) = suboptimal level of NO 3 - (773  M) in medium, HN (high nitrate) = optimal level of NO 3 - (1158  M) in medium. Columns characterize nitrate content in  mol NO 3 - / g leaf fresh weight. NITRATE UPTAKE OF WILD TYPE AND TRANSGENIC P SAG 12 -IPT WHEAT CV. SCAMP Time [min] Nitrate content in medium [  mol] controlP SAG 12 -ipt Figure 4. control = wild type wheat, P SAG 12 – IPT = ipt transformed plants with senescence inducible promotor, Points characterize nitrate content in medium in  mol et time. nitrate levelactivityspecific activity [pmol Ade / h x g FW][nmol Ade / mg prot. x h] controlLN0,03060,0086 HN0,01460,0020 P SAG 12 - IPTLN0,00570,0086 HN0,01470,0082 CYTOKININ OXIDASE/DEHYDROGENASE ACTIVITY IN WILD TYPE AND TRANSGENIC P SAG 12 -IPT WHEAT CV. SCAMP Table 2: control = wild type wheat, P SAG 12 –IPT = ipt transformed plants with senescence inducible promotor, LN (low nitrate) = suboptimal level of NO 3 - (773  M) in medium, HN (high nitrate) = optimal level of NO 3 - (1158  M) in medium, Ade = adenine, FW = fresh weight. The aim COMPARISON OF * THE PROGRESS OF SENESCENCE CYTOKININ CONTENT AND CYTOKININ OXIDASE/DEHYDROGENASE ACTIVITY NITRATE CONTENT AND NITRATE REDUCTASE ACTIVITY THE RATE OF NITRATE UPTAKE IN THE 1 ST AND 2 ND LEAF OF THE WILD-TYPE AND P SAG 12 – IPT TRANSGENIC WHEAT CV. SCAMP PLANTS AT OPTIMAL ( 1158  M) AND SUBOPTIMAL (773  M) NITRATE LEVEL. Conclusions THE SENESCENCE OF TRANSGENIC P SAG 12 -IPT PLANTS WAS DELAYED, MAINLY IN THE FLAG LEAF (FIG. 1). MAJORITY OF PHYSIOLOGICALLY ACTIVE CYTOKININS IN TRANSGENIC P SAG 12 -IPT PLANTS WAS CONVERTED TO STORAGE AND INACTIVE DERIVATES INCLUDING O- AND N-GLUCOSIDES AND CIS DERIVATES OF ZEATIN. OPTIMAL LEVEL OF NITRATES IN MEDIUM INCREASED THE CONVERSION OF ACTIVE CYTOKININS TO INACTIVE N-GLUCOSIDES IN CONTROLS (TABLE 1). CYTOKININ OXIDASE/DEHYDROGENASE ACTIVITY AND TOTAL PROTEIN CONTENT WERE HIGHER IN CONTROLS COMPARE TO TRANSGENIC P SAG 12 -IPT PLANTS; OPTIMAL LEVEL OF NITRATES DECREASED CKX ACTIVITY IN CONTROLS (TABLE 2). LEAVES OF CONTROL PLANTS SHOWED HIGHER LEVEL OF NITRATE AND SIMILARY HIGHER ACTIVITY OF NITRATE REDUCTASE COMPARED TO TRANSGENIC P SAG 12 –IPT WHEAT (FIG. 2 AND 3). NITRATE UPTAKE ET ANTHESIS WAS HIGHER AND MORE EXTENDED IN TRANSGENIC P SAG 12 -IPT PLANTS COMPARE TO CONTOLS (FIG. 4). References AOAC Official Methods of Analysis: Chlorofhyll in plants. Spectrophotometric metod for total chlorophyl and the a and b components, pp 62 - 63 (1990) Dobrev P., Kaminek M. (2002). J. Chromat. A 950:21-29 Motyka V., Faiss M., Strnad M., Kamínek M., Schmülling T. (1996). Plant. Physiol. 112: 1035-1043. Yoshida A. S. (2003). Curent Opinion in Plant Biology 6, 79-84 Knowledges This research was supported by the Grant Agency of the Czech Republic (grant No. 522/02/0530).