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JXB Advance Access published online on February 10, 2008
Journal of Experimental Botany, doi:10.1093/jxb/erm319
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© 2008 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)

RESEARCH PAPER

Senescence-induced ectopic expression of the A. tumefaciens ipt gene in wheat delays leaf senescence, increases cytokinin content, nitrate influx, and nitrate reductase activity, but does not affect grain yield

Blanka Sykorová1, Gabriela Kuresová2, Sasha Daskalova3,*, Marie Trcková2, Klára Hoyerová1, Ivana Raimanová2, Václav Motyka1, Alena Trávnícková1, Malcolm C. Elliott3 and Miroslav Kamínek1,{dagger}

1Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Rozvojová 263, 165 02 Prague 6, Czech Republic
2Crop Research Institute, Drnovská 507, 161 00 Prague 6, Czech Republic
3Norman Borlaug Institute for Plant Science Research, De Montfort University, Leicester LE7 9SU, UK

{dagger} To whom correspondence should be addressed. E-mail: kaminek{at}ueb.cas.cz

Received 3 September 2007; Revised 20 November 2007 Accepted 22 November 2007


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
The manipulation of cytokinin levels by senescence-regulated expression of the Agrobacterium tumefaciens ipt gene through its control by the Arabidopsis SAG12 (senescence-associated gene 12) promoter is an efficient tool for the prolongation of leaf photosynthetic activity which potentially can affect plant productivity. In the present study, the efficiency of this approach was tested on wheat (Triticum aestivum L.)—a monocarpic plant characterized by a fast switch from vegetative to reproductive growth, and rapid translocation of metabolites from leaves to developing grains after anthesis. When compared with the wild-type (WT) control plants, the SAG12::ipt wheat plants exhibited delayed chlorophyll degradation only when grown under limited nitrogen (N) supply. Ten days after anthesis the content of chlorophyll and bioactive cytokinins of the first (flag) leaf of the transgenic plants was 32% and 65% higher, respectively, than that of the control. There was a progressive increase in nitrate influx and nitrate reductase activity. However, the SAG12::ipt and the WT plants did not show differences in yield-related parameters including number of grains and grain weight. These results suggest that the delay of leaf senescence in wheat also delays the translocation of metabolites from leaves to developing grains, as indicated by higher accumulation of (15N-labelled) N in spikes of control compared with transgenic plants prior to anthesis. This delay interferes with the wheat reproductive strategy that is based on a fast programmed translocation of metabolites from the senescing leaves to the reproductive sinks shortly after anthesis.

Key words: Cytokinins, grain yield, ipt gene, nitrate, nitrate reductase, SAG12 promoter, senescence, wheat


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Leaf senescence is a programmed process representing the final phase of leaf development. In addition to chloroplast disintegration, protein and nucleic acid degradation, and a decline in photosynthesis, it also includes mobilization and recycling of nutrients and organic resources from senescing leaves to young plant organs (Thomas and Stoddart, 1980; Gan and Amasino, 1995; Buchanan-Wollaston, 1997; Himelblau and Amasino, 2001), thereby preventing them from premature death (Wingler et al., 2005).

Senescence is an active, energy-requiring process (Noodén, 1988) that involves preferential expression of senescence-associated genes (SAGs) (Lohman et al., 1994; Buchanan-Wollaston, 1997; Quirino et al., 2000). Like many other developmental processes, it is, at least in part, under hormonal control. While ethylene, abscisic acid, and methyl jasmonate promote leaf and cotyledon senescence (Ueda et al., 1981; Noodén, 1988; Naik et al., 2002; Ananieva et al., 2004), cytokinins delay senescence-associated processes. These include degradation of chlorophyll, chloroplast proteins (Van Staden et al., 1988; Jordi et al., 2000; McCabe et al., 2001; Chang et al., 2003), and enzymes involved in photosynthetic metabolism such as NADH-dependent hydroxypyruvate reductase and ribulose-1,5-biphosphate carboxylase/oxygenase (Wingler et al., 1998).

Gan and Amasino (1995) were the first to combine a promoter of a SAG, specifically Arabidopsis SAG12 promoter, with the Agrobacterium isopentenyl transferase (ipt) gene and generate transgenic tobacco plants with an autoregulated system for senescence-controlled cytokinin biosynthesis leading to a prolonged photosynthetic life span. This approach was later successfully applied to delay the process of senescence in a variety of plant species (Chen et al., 2001; McCabe et al., 2001; Lin et al., 2002; Chang et al., 2003; Cowan et al., 2005; Swartzberg et al., 2006).

Senescence can be triggered by a high availability of carbon relative to nitrogen (N), e.g. glucose in combination with a low N (LN) supply can induce yellowing and alter, in a senescence-specific manner, gene expression in Arabidopsis leaves (Pourtau et al., 2004, 2006). This indicates that plants grown under LN supply may respond more readily to an increase in cytokinin levels achieved by exogenous cytokinin application or ectopic ipt gene expression. Experiments performed with tobacco, wheat, and maize support this conclusion (Jordi et al., 2000; Kamínek et al., 2003; Robson et al., 2004). Thus, only wheat plants grown under LN supply, but not those kept in nutrient solution containing near optimum concentration of N (ON), showed a higher rate of net NO3– uptake following cytokinin application at the beginning of the intensive stem elongation phase (Trcková and Kamínek, 2000). The transgenic SAG12::ipt tobacco plants supplied with LN typically have an inverted N profile caused by preferential allocation of N to the old senescing leaves. At the same time, the N content of the young leaves, which serve as the main source of N and other assimilates for developing seeds, was significantly reduced (Jordi et al., 2000). Nevertheless, the total accumulation of dry mass and the seed yield were increased (Gan and Amasino, 1995).

Extensive analysis of the relationship between photosynthesis and yield in different plant species has pointed to the feasibility of a strategy aimed at improving seed yield by enhancement of single-leaf photosynthesis (Nelson, 1988). In this context, it seems reasonable to assume that expression of the ipt gene under the control of the SAG12 promoter, which leads to a delay in foliar senescence (Gan and Amasino, 1995), could increase plant productivity. On the other hand, such an approach, especially in the case of annual monocarpic crop plants characterized by an abrupt leaf senescence following initiation of seed development, may interfere with their reproductive strategy. This strategy is based on fast translocation of metabolites from the senescing leaves, where the majority of relatively easily accessible N is found (Hörtensteiner, 2006), to the developing reproductive sinks (Noodén, 1988; Miceli et al., 1995).

Wheat (Triticum aestivum L.), like other cereals has adopted a strategy of switching abruptly from vegetative to reproductive growth shortly after pollination (Humbeck et al., 1996; Trcková and Kamínek, 2000). The objective of this study was to investigate the effects of senescence-induced autoregulated ipt expression in stably transformed SAG12::ipt wheat plants on leaf senescence and cytokinin levels. Their impact on the physiological processes associated with cessation of vegetative growth and on grain yield-related parameters was also studied. It is reported that delayed leaf senescence in wheat hampers the translocation of metabolites from leaves to developing grains and thus interferes with the wheat reproductive strategy.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Construction of SAG12::ipt plants
Wheat plants (T. aestivum L. cv. Scamp) were grown in a greenhouse at 22 °C/16 °C and with a 16 h light/8 h dark photoperiod. Immature embryos were excised from the surface-sterilized seeds (10% Domestos for 20 min, five washes with sterile distilled water) 15 days after anthesis (DAA) under aseptic conditions and cultivated for a week as described by Becker et al. (1994).

Transformation was performed by a direct gene delivery method using a BioRad PDS 1000/He biolistic particle delivery system and a protocol described by the manufacturer. The plasmid pDB1 containing the uidA gene under control of the actin1 promoter and the bar gene under control of the cauliflower mosaic virus (CaMV) 35S promoter (Becker et al., 1994) was delivered together with the pSG516 plasmid comprising the Arabidopsis thaliana SAG12 promoter and Agrobacterium tumefaciens ipt gene (Gan and Amasino, 1995; Fig. 1). The bombarded explants were cultured for shoot and root regeneration as described by Becker et al. (1994) in the presence of bialaphos (4 mg l–1 during shoot formation and 1 mg l–1 during plantlet formation). The plantlets were transferred to soil and grown in the greenhouse conditions described above.


Figure 1
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  		Fig. 1. Schematic representation of the plasmids used in transformation experiments. SAG12, promoter of the A. thaliana SAG12 gene; actin1, O. sativa L. actin1 promoter; 35S, cauliflower mosaic virus 35S promoter; nos, Agrobacterium tumefaciens nopaline synthase gene terminator; ipt, A. tumefaciens isopentenyl transferase gene; uidA, Escherichia coli β-D-glucuronidase gene; bar, phosphinothricin acetyl transferase gene.

 
Genomic DNA from the leaves of the primary transformants was isolated by the method of Michaels et al. (1994), digested with PstI (an enzyme which has two restriction sites flanking the SAG12::ipt nos cassette) and subjected to electrophoresis on 0.8% agarose gels. Transfer to a Hybond N-membrane and hybridization with a [32P]dCTP-labelled (Rediprime II kit, Amersham Pharmacia Biotech, Uppsala, Sweden) ipt probe (0.7 kb NcoI/EcoRI fragment of pSG516) were carried out according to standard procedures (Sambrook et al., 1989). A Universal Genome Walker Kit (Clontech, Palo Alto, CA, USA) was used to gain information about the transgene integration sites. The sequences of the designed specific primers designated for the primary and the secondary PCR were 5'-GAACGTAGATTGTTATGGGTTCTTCTAATG-3' and 5'-AACAGACTCGGTGCTCCACGAGAATAGT-3', respectively.

Total RNA was extracted by the method of Verwoerd et al. (1989). A total of 20 µg was loaded on 1.2% formaldehyde gels, blotted onto a nylon membrane, and hybridized at 65 °C overnight with the ipt probe prepared as described above. Blots were washed twice with 2x SSC, 0.1% SDS at 65 °C for 5 min, twice with 0.1x SSC, 0.1% SDS at 65 °C for 15 min, and exposed to Kodak XAR 5 film at –70 °C. Reverse transcription-PCR (RT-PCR) was performed with a Qiagen OneStep RT PCR Kit (Qiagen, Hilden, Germany) using 1 µg of total RNA (SV Total RNA Isolation System, Promega, Southampton, UK) and the 5'-CGTCTAATTTTCGGTCCAAC-3'/5'-AGGGAATTTCTGTTCTTGTCG-3' set of primers.

The presence of the ipt gene in the T2 progeny was checked by PCR. Genomic DNA was extracted from the second leaf shortly after its appearance using a Plant DNAzol kit (Invitrogen, Carlsbad, CA, USA). PCR was performed with the ipt-For (5'-GACGCAAATATGGAAGGTAAGT-3')/ipt-Rev (5'-GAATTTCTGTTCCTGTTG-3') pair of primers. Transgene transcripts were detected by RT-PCR performed according to the manufacturer's protocol (RNeasy Plant Kit, Oligotex mRNA Kit, Omniscript RT Kit, Qiagen, Hilden, Germany).

Hydroponic cultivation of transgenic plants
The SAG12::ipt and WT wheat plants selected for further experiments were grown hydroponically in a growth room at 21 °C/15 °C and a 16 h light/8 h dark photoperiod (photon flux of 400 µmol m–2 s–1) using continuously aerated nutrient solution with 773 µM N (LN) containing Ca(NO3)2 (316 µM), KNO3 (141 µM), KH2PO4 (105 µM), MgSO4 (82.5 µM), KCl (95 µM), H3BO3 (2.5 µM), Fe-EDTA (2 µM), ZnSO4 (0.2 µM), MnSO4 (0.2 µM), CuSO4 (0.05 µM), and (NH4)6Mo7O24 (0.01 µM). In the experiment aimed at comparing the level of chlorophyll in the leaves of WT and SAG12::ipt plants, a nutrient solution containing near optimum concentration of N (1158 µM N; ON) was used. The solutions were changed weekly, and the concentration of nutrients was controlled and adjusted each second day.

Determination of chlorophyll content
Frozen leaves were homogenized in liquid nitrogen and extracted overnight in darkness with cold (4 °C) dimethylformamide according to the protocol of Lichtenthaler (1987). The absorbance at A647, A664, and A750 was measured on a Unicam 5625 spectrometer and the chlorophyll content was calculated as described by Lichtenthaler (1987).

Analysis of cytokinins
Endogenous cytokinins were extracted by methanol/formic acid/water (15:1:4, v/v/v), homogenized in liquid nitrogen, and purified using the dual-mode solid phase extraction method (Dobrev and Kaminek, 2002). Cytokinin ribotides were determined as corresponding ribosides following their dephosphorylation by alkaline phosphatase. Detection and quantification were carried out using HPLC/MS (Finnigan, San Jose, CA, USA) operated in the positive ion full-scan MS/MS mode using a multilevel calibration graph with 2H-labelled cytokinins as internal standards. Detection limits of different cytokinins were between 0.5 pmol and 1.0 pmol per sample. Results represent averages of analyses from three independent samples and two HPLC MS/MS injections for each sample.

Nitrate influx and nitrogen partition
A net NO3– influx was measured in depletion experiments. Depending on the stage of development and plant size, each selected intact plant was transferred into 300–1000 ml of well-aerated fresh nutrient solution (250 µM NO3–). After a lag period of 30 min, 5 ml samples of the nutrient solution were collected at 30 min intervals. The NO3– concentration was determined after reduction to NO2– by passing through a granulated copper–cadmium column. The nitrite was estimated spectrometrically by measuring the conversion of sulphanilamide and {alpha}-naphthylethylenediamine dihydrochloride to azo dye at 540 nm using a Skalar San plus analyser (Breda, The Netherlands). The net NO3– influx was determined from the dynamics of NO3– depletion and expressed as µmol plant–1 h–1.

For measuring the current N partitioning, selected plants at the stage of the flag leaf sheath extension (DC 41 stage of development as defined by Tottman and Makepeace, 1979) were transferred for 24 h into a well-aerated complete nutrient solution supplied with 46.8 µM 15NO3– [3.9 mg of Ca (NO3)2 per plant]. When the feeding ended, the roots were rinsed with nutrient solution containing unlabelled N and plants were transferred back into the fresh nutrient solution. The 15N-labelled plants were harvested either 4 d after the end of feeding or at the stage of full maturity. Shoots and roots were separately weighed, dried, and ground to a fine powder. Total N and 15N content of the samples was determined using EA Eurovector–Micromass IRMS IsoPrime (EuroVector, Milan, Italy). The amount of 15N allocated to different plant parts was expressed as a percentage of absorbed N.

Determination of nitrate reductase activity
Leaf and root samples [1 g fresh weight (FW)] were homogenized in liquid nitrogen and extracted with 5 ml of 50 mM TRIS-HCl buffer (pH 8.0) containing 3% (w/v) bovine serum albumin at 4 °C for 30 min. The insoluble material was removed by centrifugation (15 000 g, 30 min). The nitrate reductase (NR) activity was determined by an in vitro assay as described by Gaudinová et al. (1990). The reaction mixture consisted of 0.5 ml of 0.1 M phosphate buffer (pH 7.5), 0.1 M KNO3, 0.15 ml of enzyme extract, and 0.15 ml of 0.2% (w/v) NADH+. After incubation for 10 min at 25 °C, the reaction was terminated by addition of 0.1 ml of 0.03 M oxaloacetic acid. NR activity was expressed as the rate of generated NO2– (nmol g–1 FW min–1).

Statistical analysis
All experiments were performed three times with 2–4 independent samples each time. Means of one representative experiment are presented. The statistical significance of differences in the mean values of the examined parameters between the SAG12::ipt and WT plants was determined using the t-test (P <0.05).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Molecular analyses of SAG12::ipt wheat plants
Three independent wheat regenerants were recovered after direct gene co-delivery of pSG516 and pDB1 plasmids. Southern blot analysis using genomic DNA digested with PstI to release the 3.18 kb SAG12::ipt.nos fragment demonstrated that all primary transformants had at least one intact ipt expression cassette. Plants SI/2 and SI/3 showed additional larger hybridizing bands, indicating that, along with the intact copies there were some rearranged copies. Only one plant (SI/1) showed a single band of the expected size (Fig. 2a). This plant had the highest level of ipt expression according to RT-PCR (Fig. 2c) and northern blot (Fig. 2d) analyses. An attempt was made to obtain more information about the insertion sites of the transgene copies of this plant by DNA walking (Clontech Universal GenomeWalker Kit), but it was not possible to relate the read sequences to the annotated wheat genome sequences. However, the detection of four bands after the secondary PCR with all four genomic DNA libraries indicated the presence of at least four copies of the transgene (Fig. 2b).


Figure 2
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  		Fig. 2. Molecular analyses of the primary SAG12::ipt wheat tranformants SI1, SI2, and SI3. (a) Southern blot analysis using genomic DNA digested with PstI to release the 3.18 kb SAG12::ipt.nos expression cassette. The asterisk denotes the position of the 3.18 kb fragment. (b) Products of GenomeWalker secondary PCR with DraI (DL1), EcoRV (DL2), PvuII (DL3), and StuI (DL4) genomic DNA libraries. (c) RT-PCR of SI1, SI2, and SI3 primary transformants. An asterisk denotes the position of the expected RT-PCR product. M, lambda DNA HindIII markers. (d) Northern blot using 20 µg of total RNA of each SI1, SI2, and SI3 primary tranformant. Total RNA used for RT-PCR and northern blot analyses was isolated from flag leaves at 14 DAA.

 
Plants SI/1 and SI/3 exhibited delayed leaf senescence as indicated by higher chlorophyll content (32% and 25%, respectively) and contained higher levels of bioactive cytokinin bases and ribosides (65% and 42%, respectively) in the first leaf of the main stem compared with the control. One of the plants (SI/1) that produced enough seeds was selected for further experiments where, in addition to chlorophyll and cytokinin levels, the nitrate influx, NR activity, and current N partitioning were also determined. The presence of the ipt transgene and its expression in the T2 generation of this plant was confirmed by PCR and RT-PCR, respectively. A band of 650 bp of the ipt gene was detected in the transgenics, but not in the control plants. Representative results with plants of the T2 progeny are shown in Fig. 3.


Figure 3
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  		Fig. 3. Representative PCR (a) and RT-PCR (b) analyses of the first leaves of T2 SAG12::ipt wheat plants. k+, positive control (plasmid DNA); k–, negative control (no template); k, negative control (non-transgenic plant DNA); 27, 28, individual plants of the T2 progeny of SAG12::ipt wheat. A Fermentas GeneRuler DNA Ladder SM0331 (10–10 000 bp) was used as a marker. Arrows denote the position of the expected PCR/RT-PCR product.

 
Chlorophyll content of SAG12::ipt wheat plants grown under LN and ON supply
A decline in chlorophyll content in leaves was used as a criterion of leaf senescence. The content of chlorophyll a and b of the SAG12::ipt and WT plants grown under ON and LN supply was measured 10 DAA. Differences in chlorophyll content were found only when plants were grown under LN supply. The chlorophyll content of the first (flag) leaves of transgenics was 32% higher than the controls, while there was only a 10% increase for the second leaves. The first leaves of the main stem of both SAG12::ipt and WT plants contained more chlorophyll than the corresponding second leaves, regardless of N levels. The chlorophyll a/b ratio showed no statistically significant difference and was in the range 1.8–2.1 (Fig. 4).


Figure 4
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  		Fig. 4. Chlorophyll content of the first and the second leaves of the main stems and tillers of the SAG12::ipt and control (WT) wheat plants at 10 DAA. Plants were grown in nutrient solution containing either low or near optimum concentrations of NO3– (773 µM and 1158 µM NO3–, respectively). Error bars represent the SE (n=3).

 
Alterations in cytokinin content of SAG12::ipt transgenic wheat
Differences in chlorophyll content of the leaves of SAG12::ipt and WT wheat plants grown under LN supply were correlated to the levels of cytokinins. Twenty-two different cytokinins were identified in the first and second leaves in amounts allowing their quantification. For easy functional interpretation, the analysed cytokinins were divided according to their structure and biological activity into five groups: (i) bioactive cytokinin bases and ribosides that exhibit high activity in bioassays (Skoog and Ghani, 1981) and are recognized (most of them) by cytokinin receptors (Spíchal et al., 2004; Yonekura-Sakakibara et al., 2004); (ii) storage O-glucosides (Letham et al., 1983); (iii) irreversibly inactive N-glucosides (Letham et al., 1983); (iv) ribotides, representing the first products of cytokinin biosynthesis that were determined after the enzymatic hydrolysis to corresponding ribosides; and (v) cis-zeatins that exhibit very low cytokinin activity (Kamínek et al., 1979; Skoog and Ghani, 1981).

The leaves of both transgenic and control plants contained 5- to 15-fold higher amounts of cis-zeatin-type cytokinins compared with the amount of all other cytokinins taken together (Fig. 5). At 2 DAA the levels of different cytokinins in the transgenics and the controls were still not statistically different (results not shown). However, at 10 DAA, besides cis-zeatins, the sum of all other cytokinins in the first and the second leaves of the main stem of the SAG12::ipt plants increased by 40% and 11%, respectively, compared with the corresponding WT controls. The bioactive cytokinin bases and ribosides were impacted to the greatest extent, showing an increase of 65% and 10% over the control first and second leaves, respectively (Fig. 5). Similarly, the content of the storage forms of bioactive cytokinins, the cytokinin O-glucosides, was increased by 40% in the first leaf of the main stem of SAG12::ipt plants. The first leaves of transgenic plants also showed higher contents of cytokinin ribotides and inactive cytokinin N-glucosides, but the differences were not statistically significant compared with the controls.


Figure 5
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  		Fig. 5. Cytokinin content of the first and the second leaves of the main stems (a) and tillers (b) of the SAG12:ipt and control (WT) wheat plants at 10 DAA. Plants were grown in nutrient solution containing a suboptimum concentration of NO3– (773 µM). B+R, sum of cytokinin bases and ribosides of all cytokinins except cis-zeatins; O-G, cytokinin O-glucosides; N-G, cytokinin 7- and 9-glucosides; cis-Zs, the sum of cis-zeatin derivatives. Error bars represent the SE (n=3).

 
Nitrate influx, 15N allocation, and NR activity of SAG12::ipt wheat plants
Cessation of vegetative growth and leaf senescence are associated with a decline in the uptake of nutrients, namely of N. The delay of leaf senescence in SAG12::ipt plants was reflected in the higher, but statistically insignificant, influx of nitrate per plant already at the stage of rapid flag leaf sheet extension (D42), and at anthesis (0 DAA) compared with the control. The difference in N influx between the SAG12::ipt and WT plants progressively increased during grain formation, resulting in statistically significant 85% and 9-fold rises over the WT at 15 and 30 DAA, respectively (Fig. 6). Similar differences were found when the nitrate influx was expressed per gram FW.


Figure 6
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  		Fig. 6. Nitrate influx of SAG12::ipt and control (WT) wheat plants at different stages of development calculated from the kinetics of N depletion from the nutrient solution containing 250 µM NO3–. Error bars represent the SE (n=3).

 
NR activity known to be induced by both nitrate and cytokinins was 37% higher in the flag leaf of the SAG12::ipt plants at anthesis compared with the control values; at 10 DAA this difference increased to 80%. Older leaves from transgenic and WT plants had similar activities (Fig. 7). The current N partitioning expressed as a portion of 15N taken up by different plant parts prior to anthesis at the stage of visibly swollen boots (DC42) was 21% higher in spikes and 18% lower in the first leaf of control compared with transgenic plants. These results indicated a delay of N translocation from the leaves to the reproductive structures of transgenic plants. The differences in current N partitioning between the grains and leaves of matured control and transgenic plants were not statistically significant (Fig. 8).


Figure 7
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  		Fig. 7. Nitrate reductase activity (NRA) of different parts of the SAG12::ipt and control (WT) wheat plants at DC43 and maturity stage. Error bars represent the SE (n=3).

 

Figure 8
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  		Fig. 8. Current N partitioning in the SAG12::ipt and control (WT) wheat plants following 24 h feeding with complete nutrient solution supplemented with 15N shortly before anthesis (DC43 stage). Error bars represent the SE (n=3).

 
Grain yield components and phenotype of SAG12::ipt wheat plants
No statistically significant differences in analysed grain yield components were found between the SAG12::ipt and WT plants, including the number of spikelets per spike (25.3 and 24.7, respectively), number of grains per spike (52.4 and 54.1, respectively), weight of spikes (2.6 g and 2.9 g, respectively), grain weight per spike (2.1 g and 2.2 g, respectively), grain weight (38.9 mg and 41.1 mg, respectively), and number of tillers (7.8 and 6.7, respectively).

The delayed senescence trait was associated with minor changes in SAG12::ipt plant morphology and development. Flowering of the progeny of both transgenic lines was delayed by 3–6 d. Plants of the T2 generation of SI/1 transformants formed a slightly higher number of spikelets (by 5%) and were more susceptible to powdery mildew.


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Grains of monocarpic plants at a very early stage of their development appear to generate strong signal(s) for the onset of leaf senescence and for the recycling of nutrients and metabolites from the leaves to the reproductive organs. For example, in barley, the photosynthetic leaf capacity shows lower levels as early as 8 DAA. This is accompanied by a decrease in chlorophyll content, photosystem II efficiency, and levels of cytochrome f and the large Rubisco subunit (Humbeck et al., 1996). In the present study, no visible symptoms of senescence were detected at anthesis. Ten days later, the control plants had significantly lower chlorophyll content than the SAG12::ipt plants. However, differences between the control and the experimental groups were observed only when plants were grown under LN conditions. Similarly, the stay-green phenotype of the transgenic maize plants expressing the ipt gene under the control of the endogenous senescence-enhanced SEE-1 promoter was distinguishable from the controls only at low nutrient regimes (Robson et al., 2004). These results could be explained by the fact that low N availability accelerates the progress of senescence (Jordi et al., 2000), affects plant cytokinin status (Sattelmacher and Marschner, 1978; Takei et al., 2001, 2002; reviewed in Sakakibara et al., 2006), and reduces cytokinin delivery from roots to shoots via the xylem (Samuelson et al., 1992; Wagner and Beck, 1993; Rahayu et al., 2005).

One would expect that due to the senescence-triggered ipt expression, the preservation of cytokinin levels in the older second leaf of the SAG12::ipt wheat plants should be higher than that in the younger first leaf. However, despite the initial differences in the cytokinin content between these two leaves, the ectopic ipt expression affected them in a similar way (compare Fig. 5a and b). A dominancy of the flag leaf with regard to the supply of the plant with photosynthates (Inoue et al., 2004) and/or the loss of function of the SAG12::ipt cassette in the rapidly ageing and dying older wheat leaves could be pointed out as possible reasons for the observed phenomenon.

Similarly to Robson et al. (2004) who reported relatively minor changes in the morphology of SEE1::ipt maize plants, major morphological differences between the SAG12::ipt and control wheat plants were not detected. Expression of the ipt gene under the control of different SAG promoters in lettuce and broccoli, although causing a significant delay of post-harvest leaf senescence, also had little effect on plant morphology (Chen et al., 2001; McCabe et al., 2001). The side-effects included delays in bolting and, similarly to wheat, in flowering. These results indicate that overproduction of cytokinins in the corresponding transgenic plants was strictly localized to leaves.

Analysis of the cytokinin content in different shoot zones of SAG12::ipt tobacco plants demonstrated that the effect of ipt expression is not restricted to the old senescing leaves, and thus the presence of the transgene does not reverse the base to apical cytokinin gradient (Cowan et al., 2005). The ability of young leaves to synthesize their own cytokinins (Smart et al., 1991; Miyawaki et al., 2004) may also hamper the reversion of the cytokinin gradient in the senescence-triggered ipt-expressing plants. Moreover, ipt expression controlled by certain senescence-specific promoters, such as maize SEE1, could be extended to younger, expanding leaves (Robson et al., 2004).

Interestingly, the increase in cytokinin content of the SAG12::ipt wheat leaves was represented mainly by accumulation of bioactive cytokinin bases and ribosides and storage O-glucosides, suggesting increased rates of their biosynthesis, and/or reduced irreversible degradation by cytokinin oxidase/dehydrogenase, and/or inactivation by N-glucosylation.

The effect of ipt expression on cytokinin levels of SAG12::ipt wheat was not as prominent compared with some SAG12::ipt-transformed dicotyledonous plant species such as tobacco, Arabidopsis, and lettuce (Jordi et al., 2000; McCabe et al., 2001; Cowan et al., 2005; Huynh et al., 2005). A possible reason might be that the Arabidopsis SAG12 promoter does not function at its fullest capacity in a heterologous monocotyledonous environment.

The levels of cis-zeatin-type cytokinins, which exhibit low cytokinin activity (Kamínek et al., 1979; Skoog and Ghani, 1981), were similar in first and second leaves of the transgenic SAG12::ipt and control wheat plants (Fig. 5), suggesting that they are not affected by the ipt expression. In contrast, the SAG12::ipt tobacco leaves had lower cis-zeatin content and a profound gradient of cis-zeatins increasing from the bottom to the apical zone. Comparably, the WT leaves had an evenly distributed and higher cis-zeatin content (Cowan et al., 2005).

The nitrate influx before anthesis was higher in the SAG::ipt wheat than in the WT control. The difference became more profound at 15 DAA and even greater at 30 DAA, indicating that the SAG12::ipt imposing delay of leaf senescence also delayed the nitrate uptake decline (Fig. 6). According to previous results, the net NO3– uptake in wheat plants sharply drops after anthesis, and exogenous application of cytokinins at anthesis in combination with limited N availability increases NO3– uptake rates and overall plant uptake capacity significantly (Trcková and Kamínek, 2000). The delay of leaf senescence due to ipt expression may increase the sugar availability to roots, and thus may indirectly support the energy-dependent active uptake of nitrate in SAG12::ipt plants. This effect can be further enhanced by the reported repression of SAG12 and one other senescence-associated genes (sen1) by sugars (Chung et al., 1997; Noh and Amasino, 1999).

NR activity is known to be induced independently by both NO3– and cytokinins (Borris, 1967; Kende and Shen, 1972). Regulation of NR activity by endogenous cytokinin levels appears to reflect the plant developmental changes (Banowetz, 1992). Exogenous cytokinin application could also enhance NR activity, as was shown in wheat and barley seedlings (Gaudinová, 1990; Trcková and Kamínek, 2000). The present experiments demonstrated that NR activity in the flag leaf of the SAG12::ipt wheat plants at anthesis could be increased by as much as 37%. Its subsequent decrease with the progress of grain development could be slowed compared with the controls, possibly as a result of the higher levels of bioactive cytokinins and nitrate uptake (Figs 6 and 7, respectively). Interestingly, the NR activities (Fig. 7) in leaves, as well as the nitrate influx of SAG12::ipt plants (Fig. 6), were already increased at anthesis, indicating that ipt was at least slightly expressed at this stage of plant development when actually the flag leaf starts to senesce. Symptoms of limited ipt expression were reported for young leaves of SAG12::ipt plants responding to water deficit stress or LN supply, which could also induce onset of senescence in young leaves (Jordi et al., 2000; Cowan et al., 2005).

The overall changes in the physiology of SAG12::ipt wheat plants did not result in improvement of grain yield parameters. In contrast, the SAG12::ipt transgenic lines of rice exhibited increased grain setting rate and number of panicles per plant compared with the controls (Lin et al., 2002). The maize transgenic lines showed an increased number of florets per spikelet (Young et al., 2004). Interestingly, the application of exogenous cytokinin to the whole aerial part of wheat plants, including the ears, increased grain number per plant (Kamínek et al., 2003). Injection feeding of maize stems under the ears with cytokinin at pollination also increased the number of grains per ear and grain yield without affecting the leaf senescence (Dietrich et al., 1995). Taken together, these data suggest distinct effects of cytokinins on leaf senescence and on formation of reproductive sinks. In leaves, cytokinins delay senescence and recirculation of nutrients and organic resources to developing grains, while when applied to or expressed in the ears they stimulate grain development (Trcková and Kamínek, 2000).

In this respect, it is interesting that the delay of leaf senescence in SAG12::ipt plants affected partitioning of the currently absorbed 15N from the [15N]O3– temporarily applied at the early stage of ear development. Lower 15N partition into the growing spikes of SAG12::ipt than in WT plants and the opposite partition ratio in corresponding flag leaves suggest partial retention of N in mature leaves at the expense of the developing spikes (Fig. 8). Thus, expression of ipt in the wheat plants may support the sink strength of the leaves for N, and possibly for C and some other macronutrients, namely P, K, and S. Enrichment of leaves from line SI/1 with organic N could, in addition to other factors, be responsible for increased susceptibility of this line to powdery mildew. Accumulation of amino acids in excised tobacco leaves on the sites of application of kinetin had already been reported in the early days of cytokinin research (Mothes et al., 1961).

A recent study of Boonman et al. (2006) showed that the performance of SAG12::ipt tobacco plants grown in competition for space with WT plants is compromised because of the greater costs of respiration in lower shaded leaves retained by the transgenic plants. Moreover, this negative effect increased with canopy growth. However, in the present experiments, the expression of the ipt gene in transgenic wheat delayed senescence of the first (flag) leaf, which is the main source of metabolites for the developing reproductive organs, while senescence of the older leaves was not significantly affected (Fig. 4). Positioning of the flag uppermost leaf in the apical region of the stem near the ear limits the effect of shading even at high canopy density. Nevertheless, retention of resources in the flag leaf suppresses their extensive utilization by developing grains and seems to be responsible for elimination of the effect of prolonged leaf photosynthetic activity on supply of metabolites to developing spikes and grains. A similar current partition of N in matured grains supports such a view (Fig. 8). Comparison of the dry weight of the main stem and tillers as well as the corresponding current 15N partition indicates that expression of the ipt gene suppresses the dominance of the main stem over the tillers (results not shown). Suppression of the main stem and enhancement of the lateral bud growth including tiller formation by cytokinins has already been reported for oats (Harrison and Kaufman, 1980), wheat (Shanahan et al., 1985), and rice (Buu and Chu, 1983).

In conclusion, the senescence-controlled expression of the ipt gene delayed leaf senescence of the SAG12::ipt wheat plants grown under LN supply. This effect was associated with higher contents of bioactive and storage cytokinins and delayed decline of nitrate influx and NR activity after anthesis. In spite of these effects, which favour extension of the period of active photosynthesis and nitrate uptake, the grain yield parameters of the transgenics were not improved. This suggests that the ipt-induced delay of leaf senescence may interfere with the developmental strategy of the wheat plants, which is based on rapid translocation of available metabolites and nutrients from the leaves, and other plant parts, to the developing grains immediately after anthesis. A transgenic approach based on specific expression of the ipt gene in grain at the early stage of grain development is likely to be a more promising strategy for increasing the grain yield. The enhancement of cytokinin accumulation in inflorescence meristems of rice by reduced expression of the cytokinin-degrading enzyme cytokinin oxidase/dehydrogenase (OsCKX2) resulted in improved grain yield due to the increase in the number of reproductive organs (Ashikari et al., 2005), and therefore supports such an opinion.


   Acknowledgements
 
The authors thank Professor Richard Amasino (University of Wisconsin, Madison, WI, USA) for providing the pSG516 vector, Dr Jan Hanus and Ing. Jirí Malbeck (Institute of Experimental Botany, Prague, Czech Republic) for the synthesis of radiolabelled cytokinins and HPLC/MS analyses of cytokinins, Sam Olsen (Arizona State University, Tempe, AZ, USA) for his helpful comments, and two anonymous referees for constructive criticism and valuable suggestions. This research was supported by grants from the Grant Agency of the Czech Republic (522/02/0530), Ministry of Education, Youth and Sports of the Czech Republic (1M06030), Ministry of Agriculture of the Czech Republic (0002700601), and Grant Agency of the Academy of Sciences of the Czech Republic (IAA600380507).


   Footnotes
 
* Present address: The Biodesign Institute at Arizona State University, Tempe, AZ 85287, USA


   Abbreviations
 
cis-Z, cis-zeatin; DAA, days after anthesis; ipt, cytokinin biosynthesis gene from Agrobacterium tumefaciens; FW, fresh weight; IPT, higher plant cytokinin biosynthesis gene; LN, low concentration of nitrogen; MS, main stem; N, nitrogen; NR, nitrate reductase; NRA, NR activity; ON, near optimum concentration of nitrogen; SAG, senescence-associated gene; WT, wild type.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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