JXB Advance Access originally published online on February 21, 2005
Journal of Experimental Botany 2005 56(414):1117-1128; doi:10.1093/jxb/eri103
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RESEARCH PAPER |
Endogenous hormones and expression of senescence-related genes in different senescent types of maize
1Soil and Fertilizer Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, PR China
2National Agricultural Research Center for Hokkaido Region, Sapporo, 062-8555, Japan
3Graduate School of Agriculture, Hokkaido University, Sapporo, 060-8589, Japan
4Creative Research Initiative ‘Sousei’ (CRIS), Hokkaido University, Sapporo, 001-0021, Japan
* To whom correspondence should be addressed. Fax: +81 11 706 4170. E-mail: mosaki{at}chem.agr.hokudai.ac.jp
Received 29 September 2004; Accepted 10 December 2004
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Abstract |
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Levels of cytokinins and abscisic acid (ABA) and the expression of senescence-related genes were investigated in two maize (Zea mays L.) cultivars of different senescence type, cv. P3845 (stay-green) and cv. Hokkou 55 (earlier senescent), in a field study. The delay in leaf senescence in P3845 was correlated with increased levels of chlorophyll and nitrogen and a higher photon-saturated photosynthetic rate (Psat). Compared with the earlier senescent Hokkou 55, P3845 showed enhanced contents of cytokinins (trans-zeatin riboside, t-ZR; dihydrozeatin riboside, DHZR; isopentenyladenosine, iPA) and reduced levels of ABA in its leaves. In roots, P3845 had increased levels of t-ZR, DHZR, and ABA, but decreased concentrations of iPA. It was concluded that a higher rate of cytokinin transport from roots to leaves contributes to the delay of senescence in P3845. By contrast, the translocation of ABA from roots to shoots may be blocked in the stay-green cultivar, which also results in retarded leaf senescence. P3845 ear leaves contained more malondialdehyde (MDA) and higher catalase (CAT) and superoxide dismutase (SOD) activities than Hokkou 55. Since the accumulation of the mRNAs for Rubisco small subunit (rbcS), phosphoenolpyruvate carboxylase (PEPC), and SOD peaked after Chl content and Psat had reached their maxima, it is speculated that when leaf senescence is initiated, Chl contents decrease first, followed by the degradation of the photosynthetic apparatus and of photosynthesis-related enzymes. See1 and See2 encode senescence-related cysteine proteases; their mRNAs were most abundant in yellowing leaves, suggesting that these proteins are involved in the process of senescence rather than its initiation. mRNAs of both genes were more abundant in Hokkou 55 than in P3845, which suggests a regulation of leaf senescence at the transcriptional level.
Key words: Abscisic acid, cytokinins, gene expression, leaf senescence, maize
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Leaf senescence is a key step in the life cycle of an annual plant. During the process, materials used to build up leaves during vegetative growth are remobilized and transported into the developing seed (Smart, 1994
; Smart et al., 1995). Conspicuous visual symptoms of leaf senescence are the loss of chlorophyll pigments (yellowing), desiccation, and eventual abscission. Cellular and molecular events contributing to these visual symptoms include chloroplast disintegration, a decline in photosynthesis, and the loss of proteins and nucleic acids (Smart, 1994; Buchanan-Wollaston, 1997; Chandlee, 2001). Additional internal symptoms of senescence are a decreased ability to accumulate proteins and nucleic acids due to enhanced degradation and/or reduced synthesis (Smart, 1994; Buchanan-Wollaston, 1997).
Leaf senescence is a major determinant of yield in many crops (Thomas, 1992). Although senescence occurs in an age-dependent manner in many species (Noodèn, 1988), its initiation and progression can be modulated by a variety of environmental factors such as temperature, mineral deficiency, and drought conditions, as well as by internal factors such as plant growth regulators (Smart, 1994; Grbic and Bleecker, 1995; Buchanan-Wollaston, 1997; Nam, 1997; Weaver et al., 1997; Dai et al., 1999). Plant hormones have been characterized most thoroughly at the molecular and physiological levels. The best evidence for hormonal involvement in the delay of leaf senescence is for the hormones cytokinins (Noodèn, 1988; Van Staden et al., 1988). Cytokinins are a structurally diverse group of N6-substituted purine derivatives capable of inducing plant cell division. Zeatin riboside (t-ZR), dihydrozeatin riboside (DHZR), and isopentenyl adenine (iPA) are the three most commonly detected physiologically active cytokinins in plants (Mok, 1994). Numerous plant developmental processes are influenced by cytokinin, including cell expansion, inhibition of leaf senescence, chloroplast development, mobilization of nutrients, and root and shoot branching (Mok, 1994). Leaf senescence is usually correlated with a decrease in cytokinin in the leaves (Buchanan-Wollaston, 1997; Noodèn et al., 1997). Exogenous application of cytokinin inhibits the degradation of chlorophyll and of proteins of the photosynthetic apparatus (Badenoch-Jones et al., 1996; He and Jin, 1999). The strategy to delay senescence involves the transgenic expression of the IPT gene from Agrobacterium, which encodes an isopentenyl transferase. Transgenic plants with elevated cytokinin levels caused by an introduced isopentenyl transferase showed delayed senescence (Smart et al., 1991; Gan and Amasino, 1995; Robson et al., 2004). By contrast, abscisic acid (ABA) is considered a senescence promoter, although evidence for an in vivo role is rather poor compared with ethylene (Zeevaart and Creelman, 1988; Noodèn, 1988; Madhu et al., 1999; Tadas et al., 1999). Foliar spraying with ABA promoted senescence in rice (Ray et al., 1983) and maize (He and Jin, 1999). However, not much is known about the changes of cytokinin and ABA levels and their relationship to the progression of leaf senescence in stay-green maize, which shows an increased leaf lifespan.
Although extensive physiological and biochemical data on whole plant leaf senescence were available for many years, the molecular events that induce and control the process have only recently been investigated in depth (Smart, 1994; Buchanan-Wollaston, 1997; Nam, 1997; Weaver et al., 1997; Ewing et al., 1998; Yap et al., 2003). There is clear evidence that leaf senescence is a tightly regulated developmental process, and that the expression of specific genes is involved (Smart, 1994; Buchanan-Wollaston, 1997; Nam, 1997; Weaver et al., 1997; Suzuki et al., 1994). Numerous enzymes have been implicated in providing essential activities for the initiation and progression of the senescence programme including proteases, nucleases, and other degradative enzymes, as well as enzymes involved in chloroplast dismantling and chlorophyll breakdown (Smart, 1994; Buchanan-Wollaston, 1997).
The fact that there are no plant mutants known which completely block all aspects of the senescence process suggests that the senescence programme is complex and regulated through multiple, interrelated but distinct signalling pathways. However, mutants that show partial defects in the overall senescence programme do exist and give rise to ‘evergreen’ or ‘stay-green’ phenotypes. Identification and characterization of senescence mutants is an important first step in identifying regulatory genes. Several mutants have been studied that impinge on certain aspects of the leaf senescence syndrome such as chlorophyll loss, or that show a delay in senescence (Thomas and Smart, 1993). Genetic variations in the timing and rate of leaf senescence also occur, and mutants with altered senescence pattern have been used to study the cellular and molecular basis of senescence (Thomas and Smart, 1993). The availability of maize lines with contrasting senescence phenotypes offers the opportunity to compare the expression of senescence genes in different genetic backgrounds. Senescence related to carbon and nitrogen contents in leaves of two lines, stay-green and earlier senescent, that are morphologically similar and do not differ developmentally except for the different lifespan of individual leaves, has been analysed (He et al., 2001, 2002a, b, 2003, 2004). However, little work has been done on the gene expression related to leaf senescence in these two different senescence types of maize.
In this study, an attempt has been made (i) to characterize changes of the levels of cytokinins (including t-ZR, DHZR, and iPA) and ABA in the leaves and roots in a stay-green hybrid of maize, and to determine how the hormonal changes affect leaf senescence; and (ii) to elucidate the molecular mechanisms of leaf senescence by the analysis of expression patterns of senescence-associated genes of maize, such as rbcL (large subunit of Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase), rbcS (small subunit of Rubisco), PEPC (phosphoenolpyruvate carboxylase), SOD (superoxide dismutase), See1 (senescence enhanced gene, encoding cysteine protease), and See2 (senescence enhanced gene, encoding cysteine protease). The expression patterns are also discussed in the context of analyses of chlorophyll (Chl) and N contents, of photon-saturated photosynthetic rate (Psat), of enzyme activities of catalase (CAT) and superoxide dismutase (SOD), and of malondialdehyde (MDA) contents.
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Materials and methods |
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Plant materials and sampling
Seeds of two maize (Zea mays L.) hybrids, a representative stay-green line (var. P3845) and a representative earlier senescent variant (var. Hokkou 55), were soaked in water for germination. Both hybrids are morphologically similar and do not differ in phenology, which have previously been used in studying physiological processes contributing to yield increase (He et al., 2002b
, 2003). The germinated seeds were sown in a field of the Graduate School of Agriculture, Hokkaido University, Sapporo, Japan (43°03' N, 141°20' E) on 21 May, 2002. The plots (40 m2) were arranged in a randomized complete block design with three replications and a plant density of 100 000 plants ha–1 (space: 0.5x0.2 m). Nitrogen (300 kg N ha–1 in total; 50 kg N ha–1 supplied as a rapid release fertilizer, and the other 250 kg N ha–1 supplied as a slow release fertilizer), phosphorous (200 kg P2O5 ha–1), and potassium (200 kg K2O ha–1) were applied as basal applications with (NH4)2SO4 or urea, superphosphate and potassium sulphate, respectively. Individual leaves (the 6th, 9th, and 12th leaf counted from the bottom) were sampled after flowering at 2-week intervals from leafing to senescence, which corresponded to 60, 76, 93, 110, and 124 d after sowing (DAS). Leaves were frozen in liquid nitrogen and stored at –80 °C for hormone analysis. For dry mass and N content determinations, portions of the leaf samples were dried at 80 °C in an air-forced oven for 72 h, weighed, and ground. Fifty plants were sampled at harvest to determine yield and yield components.
Chlorophyll and nitrogen contents determination
Chlorophyll (Chl) contents were determined as described before (He et al., 2002b). For the determination of the Chl content, 0.25 g of fresh leaves were placed in a 100 cm3 test tube; 10–15 cm3 pure methanol was added and homogenized with a polytron. The homogenate was then filtered and filled up to 100 cm3 with pure methanol. The Chl concentration in the supernatant was spectrophotometrically determined by measuring the absorbances at 652.0 and 665.2 nm for Chl a and Chl b, respectively, and calculated according to Porra et al. (1989; Chl a + Chl b content (mg ml–1)=22.12x[absorbance at 652.0 nm]+2.71x[absorbance at 665.2 nm]). Nitrogen (N) was determined using the Kjeldahl method (Hind, 1993).
Measurement of the photon-saturated net photosynthetic rate (Psat)
To determine leaf position, individual leaves were marked from the ground level up. Psat was measured in the field at photon saturation by placing the intact leaf in transparent plastic chambers of appropriate sizes connected to an infrared gas analyser (Model ADC-3, Shimadzu, Kyoto, Japan). Light of about 1500 µmol m–2 s–1 was supplied by a halogen lamp (KTS-100R, Kenko Co. Ltd., Tokyo, Japan). The air temperature, relative humidity and CO2 concentration in the chamber were 20–25 °C, 40–50%, and 350–370 g m–3, respectively.
Hormone extraction, purification and quantification
The methods for extraction and purification of t-ZR, DHZR, iPA, and ABA were modified after Degenhardt et al. (2000). About 0.2 g fresh weight of leaves and root were extracted and homogenized in 2 ml of 80% methanol (containing 40 mg l–1 butylated hydroxytoluence) and stored at –20 °C for 48 h. After centrifugation at 20 000 g for 15 min, sediments were resuspended in 1 ml 80% methanol at –20 °C for 16 h. The combined extracts were purified by passing them through C18-Sep-Pak cartridges (Waters, Milford, USA). Afterwards, samples were evaporated under vacuum to remove the organic solvent, and dissolved in 2.0 ml of TBS buffer (tris-buffered saline; 50 mM TRIS, pH 7.8, 1 mM MgCl2, 10 mM NaCl, 0.1% Tween, 0.1% gelatin). DHZR, t-ZR, iPA, and ABA contents were determined by ELISA using monoclonal antibodies (Phytodetek, Agdia, Elkhart, IN, USA) following the protocol provided by the manufacturer. As the antibodies of cytokinins also recognize free bases, nucleotides and 9N-glucosides, what was measured was the sum of free basis, ribosides, nucleotides and 9N-glucosides of corresponding cytokinins. For simplicity, the cytokinins are further called as corresponding ribosides of ZR, DHZR and iPA. In case of ABA, the method measured is the real ABA since antibodies are raised against ABA in such a way that they do not recognize the bound forms of the hormone.
Measurements of malondialdehyde (MDA) content, and the activities of catalase (CAT) and superoxide dismutase (SOD)
MDA contents were determined by the thiobarbituric acid reaction as described by Peever and Higgins (1989). One gram (FW) of the ear leaf (12th leaf) was homogenized in 5 ml 0.1% (w/v) TCA. The homogenate was centrifuged at 10 000 g for 5 min and 4 ml of 20% TCA containing 0.5% (w/v) TBA were added to 1 ml of the supernatant. The mixture was heated at 95 °C for 30 min and then quickly cooled on ice. The contents were centrifuged at 10 000 g for 15 min and absorbance of the supernatant at 532 and 600 nm was read. After subtracting the non-specific absorbance at 600 nm, the MDA concentration was determined by its extinction coefficient of 155 mM–1 cm–1.
Enzyme extract for CAT (EC 1.11.1.6
[EC]
) and SOD (EC 1.15.1.1
[EC]
) was prepared by first freezing the weighed amount of leaf samples (0.5 g) in liquid nitrogen to prevent proteolytic activity, followed by grinding with 5 ml extraction buffer (0.1 M phosphate buffer, pH 7.5, containing 0.5 mM EDTA and 1 mM ascorbic acid). Brie was centrifuged for 20 min at 15 000 g and the supernatant was used as an enzyme. The soluble protein concentration in the supernatants was determined using the method of Bradford (1976) with bovine serum albumin as standard. CAT activity was determined by following the consumption of H2O2 (extinction coefficient 39.4 mM–1 cm–1) photometrically at 240 nm for 3 min (Aebi, 1984). The reaction mixture contained 50 mM potassium phosphate buffer (pH 7.0), 10 mM H2O2 and 200 µl enzyme extract in a 3 ml volume. Superoxide dismutase activity was estimated by recording the decrease in optical density of nitroblue tetrazolium (NBT) induced by the enzyme (Dhindsa et al., 1981). Three ml of the reaction mixture contained 13 mM methionine, 75 µM nitroblue tetrazolium chloride, 0.1 mM EDTA, 50 mM phosphate buffer (pH 7.8), 50 mM sodium carbonate, and 0.1 ml enzyme solution. The reaction was started by adding 2 µM riboflavin. The reaction mixtures were illuminated for 15 min at 5000 lx (placing the test tubes under two 15 W fluorescent lamps). A complete reaction mixture without enzyme, which gave the maximal colour, served as the control. The reaction was stopped by switching off the light and putting the tubes into dark. A non-irradiated complete reaction mixture served as a blank.
Northern blot analysis
Total RNA was isolated from ear leaf tissues using the sodium dodecyl sulphate (SDS)–phenol method (Palmiter, 1974). RNA content and purity was determined by measuring the absorbance of an aliquot at 230, 260, and 280 nm. The concentration of all samples was adjusted to 1 mg ml–1 and RNA was stored at –80 °C in small aliquots until analysis.
The fluorescein-labelled DNA probes for Rubisco large subunit (rbcL), small subunit (rbcS), phosphoenolpyruvate carboxylase (PEPC), superoxide dismutase (SOD), cysteine protease1 (See1), and cysteine protease2 (See2) were prepared from PCR fragments of the maize rbcL gene (Gaut, 1992), rbcS gene (Ewing et al., 1998), PEPC gene (Lepiniec et al., 1993), SOD gene (White and Scandalios, 1988), See1 gene (Griffiths et al., 1997), and See2 gene (accession no. AJ251454), respectively, with the Gene ImagesTM labelling and detection system (Amersham Biosciences, Piscataway, NJ, USA) according to the manufacturer's instructions.
Total RNA (5 µg for rbcL and rbcS, 10 µg for PEPC, SOD, See1, and See2) was denatured in a mixture of 70% (v/v) formamide and 8% (v/v) formaldehyde, and separated on a 1% agarose gel containing 1.8% (v/v) formaldehyde. After electrophoresis, RNAs were transferred to a Hybond-N+ membrane (Amersham Biosciences). Hybridization and detection were performed using Gene ImagesTM. Prehybridization of each membrane was carried out at 65 °C for 4 h in 5x SSC, 5% (v/v) Liquid Block, 5% (w/v) dextran sulphate, and 0.1% SDS, and was followed by hybridization with adequate amounts of fluorescein-labelled probes in fresh buffer at 65 °C for over 15 h. Membranes were washed in 1x SSC, 0.1% SDS (15 min), and 0.5x SSC, 0.1% SDS (15 min) at 65 °C. Hybridized probes were detected using the chemiluminescent substrate CDP-Star after incubation of the blots with anti-fluorescein antibody conjugated to alkaline phosphatase at 37 °C for 1 h.
Statistical analysis
Standard analysis of variance techniques were used to assess the significance of the results obtained. Differences between treatment means were compared using the least significant difference (LSD). Regression analysis was performed using SPSS 10.0 (SPSS For Windows 1999, SPSS Inc, Chicago, IL, USA).
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Results |
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Chl contents in individual leaves
Chl content in the sixth leaf of P3845 increased to reach maximum detected values at 76 DAS, and, thereafter decreased, while that in Hokkou 55 declined continuously with time (Fig. 1). Leaf 6 in Hokkou 55 senesced and died before 124 DAS, so that physiological parameters including the contents of Chl, N, and endogenous hormones could not be recorded at this stage. In both hybrids, the Chl content in the 9th and 12th leaf increased after the first sampling to reach maximum detected values at 93 DAS and, thereafter, declined. The Chl contents in leaves of the stay-green hybrid P3845 exceeded those in the corresponding leaves of the earlier senescing hybrid Hokkou 55. At the last measurement, Chl contents in the 9th and 12th leaves in P3845 were 2.85 and 1.41 times greater, respectively, than those in Hokkou 55.
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N content in leaves and roots
During the period of observation, the N content in the 12th leaves of both hybrids first increased and then declined in a similar fashion as the Chl levels did, while N in the 6th and 9th leaves declined continuously until maturity. At the last measurement, N contents in the 9th and 12th leaves of P3845 were 1.37 and 1.25 times greater than those of Hokkou 55. Nitrogen in roots decreased with time in both hybrids. No distinct difference in root N between the two hybrids was detected, except for the last sampling date (Fig. 2).
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Photon-saturated net photosynthetic rate (Psat) in leaves
Both genotypes increased to the highest Psat values detected at 93 DAS in the 6th leaf and at 110 DAS in the 12th leaf. In the 9th leaf, the highest value detected was attained at 93 DAS in Hokkou 55 and at 110 DAS in P3845. The Chl peaks (Fig. 1) appeared before the major drop in Psat with the exception of leaf 9 in Hokkou 55, indicating that the efficiency of the photosynthetic apparatus is not always reflected by the amount of Chl present. Psat tended to be higher in leaves of P3845 than in leaves of Hokkou 55 at any time. At the final measurement, Psat in the 6th leaf of Hokkou 55 had dropped to close to zero, while that in P3845 still was high (Fig. 3).
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Hormone levels in leaves and roots
In both hybrids, t-ZR contents in the leaves increased slowly during the early grain filling stage (i.e. grain filling stage started at 76 DAS), reached a maximum measured at 93 DAS in the 6th and 9th leaves and 110 DAS in the 12th leaf, respectively, and decreased thereafter. The difference in t-ZR contents in the leaves at 60 DAS (early grain filling stage) was not distinct between the two hybrids. The leaves of P3845 contained more t-ZR than those of Hokkou 55 (Fig. 4). Similar patterns of t-ZR contents were found in roots of both lines.
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Time-courses of DHZR contents in leaves (Fig. 5) and roots resembled those of t-ZR in the two hybrids. DHZR levels reached maxima measured at 93 DAS and decreased thereafter. P3845 contained more DHZR than P3845.
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By contrast to t-ZR and DHZR, iPA contents in leaves increased rapidly after grain filling stage (76 DAS) and remained at the level obtained until the end of the observation period (Fig. 6). The iPA concentrations in leaves generally appeared to be higher in P3845 than in Hokkou 55. In roots, the general trend was less clear. Root iPA concentrations seemed to decline from the start of sampling with minimum values measured at 93 DAS, and then increased.
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ABA contents in the leaves of both hybrids (Fig. 7) showed peaked time-courses that resembled those found in t-ZR (Fig. 4). However, while leaf cytokinin contents had generally been higher in P3845 than in Hokkou 55, ABA appeared more highly concentrated in the leaves of Hokkou 55 (Fig. 7). Interestingly, this was not so in roots. Here, ABA levels were greater in P3845 as compared with Hokkou 55 during the whole experimental period (Fig. 8).
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MDA content and activities of CAT and SOD
In both genotypes, MDA contents increased at increasing rates up to the stage of maturity (Fig. 9A). MDA levels tended to be slightly higher in P3845 than in Hokkou 55. By contrast, CAT activity declined with leaf age (Fig. 9B). Initially, Hokkou 55 showed higher CAT activities than P3845, but this relationship reversed as the plants matured. SOD activity increased to a maximum measured at 76 DAS in both cultivars, thereafter declined again (Fig. 9C). SOD activity was higher in Hokkou 55 than in P3845 before the maximum was reached and vice versa afterwards.
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Gene expression
Figure 10 summarizes the results of the determination of rbcL, rbcS, PEPC, SOD, See1, and See2 mRNAs as well as total RNA from ear leaves. rbcL, rbcS, PEPC, SOD, and See1 mRNAs were detectable throughout the experimental period, but See2 mRNA was only found at the last two stages.
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The levels of rbcL mRNA exhibited a gradual decrease which started before the onset of visible senescence in both lines. The levels of rbcS mRNA decreased to a minimum at 76 DAS, then increased to a maximum at 110 DAS, and finally declined. There was no obvious difference in the rbcL and rbcS mRNA levels between the two hybrids. The expression of PEPC mRNA resembled that of rbcS, but P3845 contained more PEPC mRNA than Hokkou 55 from 76 to 110 DAS. SOD mRNA increased to a peak of expression at 93 and 110 DAS in P3845 and Hokkou 55, respectively, and then dropped at maturity.
See1 and See2 mRNAs were most abundant in leaves that were visibly yellowing, i.e. after the major drop in chlorophyll content, suggesting that the encoded proteins are involved in the process of senescence rather than its initiation. The expression of both See1 and See2 showed higher levels in Hokkou 55 than in P3845.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The results of previous work suggested that leaf senescence in maize might follow a sequence leading from changes in endogenous hormone levels to transmembrane Ca2+ fluxes, which then cause lipid peroxidation and subsequent degradation of Chl and protein (He and Jin, 1999
). During senescence, endogenous signals up-regulate certain genes whose products show high homology to enzymes known to degrade protein, RNA, lipids, and chlorophyll (Buchanan-Wollaston, 1997). Although major plant hormones have been implicated in the senescence process, cytokinins and ABA have been shown conclusively to regulate senescence (Smart, 1994). Since the 9th leaf in both varieties discussed in the present paper is the ear leaf, which plays key roles in photosynthate translocation to the developing grain, the relationship of the hormone contents of this leaf and roots during late maturation (the last three sampling stages; Fig. 11) is discussed here. The roots of P3845 contained significantly higher concentrations of t-ZR than those of Hokkou 55 at 93 DAS (Fig. 8), and then decreased to almost the same level as that detected in Hokkou 55 at the last two sampling stages. Therefore, the rate of decrease of t-ZR in roots was higher in P3845 than in Hokkou 55. While P3845 contained more DHZR in both roots and shoot than Hokkou 55, the time-courses of DHZR contents were similar in roots and shoots of both varieties (Figs 5, 8). Therefore, there is no significant difference in the relationships between the DHZR contents of the 9th leaf and the roots in the two lines (Fig. 11). The relatively low iPA in roots and high iPA in the 9th leaf of P3845 caused a higher ratio of the iPA concentrations in the 9th leaf and the roots in this line (Fig. 11). It has been reported that iP-type cytokinins are considered to have low biological activity in higher plant systems (Van Staden and Drewes, 1991) and are susceptible to cytokinin oxidase degradation (Mok et al., 2000), therefore it is zeatin and not iP, which is considered to be the active form (Horgan, 1992). Cytokinin enhancement in stay-green was not as high in this study compared with the report by Singh et al. (1992) that cytokinin increased to over 1.5 times after NH4NO3 treatment. The reason for this high level of cytokinin may be due to the exogenous inducement with NH4NO3 treatment, while cytokinin differences in this study are the endogenous differences between two types of senescent types without any cytokinin or other exogenous inducement. Interestingly, P3845 showed relatively low concentrations of ABA in shoots (Fig. 7) and high concentrations in roots (Fig. 8), resulting in a lower ratio of these concentrations, compared with Hokkou 55 (Fig. 11). It was speculated that a blockage of ABA translocation from roots to shoots might play a role in the delay of senescence in P3845. If this hypothesis is the case, it would represent a novel mechanism in the regulation of senescence by plant hormones.
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It has been reported that ABA, but not cytokinins, is positively correlated with sucrose phosphate synthase (SPS) activity in both stems and leaves (Yang et al., 2002
). When ABA was applied to leaves, SPS activity increased, whereas cytokinin application had the opposite effect (Yang et al., 2002). Since ABA was positively correlated with remobilization (or translocation) of stored carbon compounds, and since such remobilization was enhanced further by exogenous ABA, it was concluded that the enhanced remobilization can be attributed, at least partly, to an elevated ABA level in leaves. On the other hand, increased cytokinin levels and reduced glucose repression of photosynthesis-related genes could also delay senescence (Quirino et al., 2000). It was reported previously that delayed senescence occurred in stay-green maize because of the lower carbon translocation rate from leaves (He et al., 2002b). Since ABA can enhance carbon remobilization or translocation (Yang et al., 2002) and cytokinins inhibit the glucose repression response (Moore et al., 2003), and since the enhanced carbon translocation correlates with earlier leaf senescence (He et al., 2002b), the stay-green phenotype seems to be explained as a result of the reduced ABA and increased cytokinin levels in these plants. Further experiments such as comparing other varieties may help in understanding the role of ABA and cytokinin.
During leaf senescence, Chl is degraded as thylakoid proteolipids are mobilized and the rate of photosynthesis declines (Thompson et al., 1987; Desimone et al., 1996; Thomas and Howarth, 2000; He et al., 2002b). In the present study, Chl contents increased up to 93 DAS (except in the 6th leaf) as the young leaf developed. Thereafter, Chl levels decreased in both lines tested, indicating the onset of senescence (Fig. 1). Importantly, the photosynthetic rate declined after the first detectable reduction in Chl contents (Fig. 2). Lipid peroxidation (as indicated by MDA contents) increased continuously with plant age (Fig. 9A), which is in line with previous reports (Hurng and Kao, 1994; Marie, 1995; Ye et al., 2000). Activities of the antioxidant enzymes CAT and SOD increased in young leaves up to 76 DAS, but decreased afterwards (Fig. 9B, C). Similar activity patterns have been found in tobacco (Dhindsa et al., 1981), Arabidopsis thaliana (Hurng and Kao, 1994), and pea (Marie, 1995). The increased activities of CAT and SOD in P3845 compared with Hokkou 55 after 76 DAS suggest that these enzymes play a role in delaying senescence in P3845. However, accumulation levels of SOD mRNA were greater in P3845 only before 93 DAS, whereas they were visibly higher in Hokkou 55 in the last two sampling stages (Fig. 10), which seems difficult to explain and needs further study. Interestingly, the peaks of the accumulation of mRNAs of rbcS, PEPC, and SOD follow the peak of Chl contents and precede the decline of CO2 fixation rate, indicating that photosynthesis-related compounds are not broken down synchronously during leaf senescence.
Since rbcL mRNA declines before the onset of visible senescence in both lines, it is possible that the Rubisco large subunit is involved in the initiation of leaf senescence. As the decline of rbcL mRNA appears correlated with the degradation of Rubisco described in a previous report (He et al., 2002b), it appears that the amount of Rubisco synthesized is primarily determined by the level of rbcL mRNA. During leaf senescence, the decomposition of Rubisco is an important component of the N-redistribution process, since in addition to their roles in photosynthetic CO2 fixation, these enzymes also constitute the major N reserve available for the developing sink (Chollet et al., 1996; Osaki et al., 1995; Rogers and Ellsworth, 2002). These results suggest that a decline of rbcL mRNA caused the degradation of Rubisco, which in turn resulted in the decrease of photosynthetic rate (Fig. 3).
See1 and See2 encode proteases that are synthesized during maize leaf senescence. See1 is specifically expressed during senescence and seed germination. See2 protease from senescing maize leaves cleaves proteins next to asparagine residues. See2 is predicted to be responsible for the activation of proteins required for senescence, which possibly includes other proteases (Kingston-Smith et al., 1999). See1 exhibited least expression in green leaves and a gradual increase in mRNA with leaf age, but See2 expressed a transient appearance only at 124 DAS, which meant that both mRNAs were most abundant in leaves that were visibly yellowing, i.e. after the major drop in chlorophyll content, suggesting that they encode proteins involved in the process of senescence rather than its initiation. Accumulation of the mRNAs of both See1 and See2 was stronger in Hokkou 55 than in P3845. It is not surprising that the level of protease mRNA should increase during senescence, as the breakdown of proteins and their remobilization to other parts of the plant is a major part of the process. A strikingly similar pattern of See1 and See2 mRNA accumulation was reported by Smart et al. (1995) in maize. These results demonstrated that the two senescence types of maize differed in the accumulation rates of senescence-related mRNAs.
In this experiment, there was no clear correlation between the N content in the ear leaf and the expression levels of See1 and See2. At maturity, the early senescing Hokkou 55 showed lower N contents and higher mRNA accumulation levels of both See1 and See2 than the stay-green variant P3845. These results suggest that high expression levels of cysteine protease are related to low N contents, and therefore to rapid leaf senescence. In plants, protein degradation is a crucial mechanism in developmental processes such as morphogenesis and cell biogenesis, senescence, and programmed cell death (Palma et al., 2003). During senescence-related N-redistribution, the concentration of soluble protein decreases in source tissues as these proteins are exported to sink organs. It is of note that leaves of stay-green maize contain more soluble protein-N than the earlier senescent types (He et al., 2001). The increased accumulation of See1 and See2 mRNAs in Hokkou 55 (Fig. 10) suggests increased rates of protease synthesis which probably leads to more efficient protein degradation and N-redistribution in this early senescence line.
It has been reported that cytokinins are involved in the expression of several senescence-related genes (Forde, 2002; Robson et al., 2004; Li et al., 2000; Martin et al., 2000; Suzuki et al., 1994; Takei et al., 2001). Li et al. (2000) reported that cytokinin delayed senescence and reduced expression of both See1 mRNA and protein. C4 PEPC gene in maize and a subgroup of genes belonging to the response regulator family in maize are rapidly induced by cytokinin treatment (Taniguchi et al., 1998; Kiba et al., 1999) The results in the current paper showed stay-green types contained more cytokinins than earlier types, and also reduced the expression of both See1 and See2 mRNAs, increased expression of PEPC mRNA, but no direct evidence existed in this paper between cytokinins and the expression of senescence-related gene, such as cysteine protease and photosynthetic related enzymes of Rubisco and PEPC, which needs further study.
In this paper, physiological and molecular parameters have been identified that have given preliminary information about the mechanisms of maize leaf senescence. Dynamic levels of phytohormones have been analysed, as well as the expression of several senescence-related genes in mature and senescing leaves of two maize lines. The accumulation patterns of transcripts of rbcL, rbcS, PEPC, and SOD during leaf senescence in maize had not been described previously. For the transcripts of See1 and See2, the patterns coincided with those reported by Smart et al. (1995). Senescence is a developmental stage that has not been studied at the level of gene expression until recently. The results presented here extend current knowledge of the gene products that are present during senescence.
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This research was supported by a fellowship from the Japan Science and Technology Agency (STA). Maize seeds were provided by Dr K Koinuma from the National Agricultural Research Center for the Hokkaido Region, Sapporo, Japan.
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